Novel ep4 agonist

ABSTRACT

Provided is a compound represented by the formula (1): 
     
       
         
         
             
             
         
       
     
     wherein R 1  and R 2  are each independently a hydrogen atom or a straight chain alkyl group having a carbon number of 1-3, R 3  is a hydrogen atom, an alkyl group having a carbon number of 1-4, an alkoxyalkyl group, an aryl group, a halogen atom or a haloalkyl group, or a pharmaceutically acceptable salt thereof, which has, unlike known PGI 2  analogs, a selective EP4 agonist action, and a medicament containing the compound, which is useful for the prophylaxis and/or treatment of immune diseases, diseases of the digestive tract, cardiovascular diseases, cardiac diseases, respiratory diseases, neurological diseases, ophthalmic diseases, renal diseases, hepatic diseases, bone diseases, skin diseases and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 14/151,479, filed Jan. 9, 2014, which is acontinuation of U.S. patent application Ser. No. 13/675,530, filed Nov.13, 2012, now U.S. Pat. No. 8,691,857, which is a continuation of U.S.patent application Ser. No. 12/917,935, filed Nov. 2, 2010, now U.S.Pat. No. 8,394,844, which is a continuation-in-part of InternationalPatent Application PCT/JP2009/065690, filed Sep. 8, 2009, and whichclaims priority to Japanese Patent Application 2009-168193, filed Jul.16, 2009, and Japanese Patent Application 2008-232133, filed Sep. 10,2008, the contents of which are encompassed in their entireties herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a 7,7-difluoroprostaglandin I₂derivative wherein the carboxy group at C-1 of prostaglandin(hereinafter to be referred to as PG) is substituted by a tetrazolegroup, and two fluorine atoms are bonded at C-7 of PG, or apharmaceutically acceptable salt thereof, a pharmaceutical compositionthereof, and a pharmaceutical use thereof. More specifically, thepresent invention relates to a 7,7-difluoroprostaglandin I₂ derivativewhich is an EP4 agonist useful for the prophylaxis or treatment ofimmune diseases, cardiovascular diseases, cardiac diseases, respiratorydiseases, ophthalmic diseases, renal diseases, hepatic diseases, bonediseases, diseases of the digestive tract, neurological diseases, skindiseases and the like.

BACKGROUND OF THE INVENTION

Natural PGs each bind to their specific receptors, and exhibitcharacteristic actions. A receptor for each of PGI₂, PGE₂, PGD₂, PGF₂αand thromboxane A₂ (TXA₂) is called IP, EP, DP, FP and TP, respectively.Furthermore, EP further has four subtypes, EP1, EP2, EP3 and EP4. ThesePG receptors show different expression patterns in the organs and cells,and even if the receptors are expressed in the same cell, the actionsshown thereby are different.

While the derivatives of the natural PGs are under influence of theoriginal carbon skeleton, they come to bind to various receptors as thestructures change (non-patent documents 1 and 2).

PG derivatives having a tetrazole group instead of the carboxy group atC-1 of prostaglandin have been reported in the following patentdocuments 1-4, non-patent document 2 and the like. Furthermore,7,7-difluoro PGI₂ analogs and manufacturing methods thereof have beenreported (patent documents 5 and 6). In addition, 7,7-difluoro PGI₂analogs are described to be useful as prophylactic or therapeutic agentsfor cardiovascular diseases (patent document 5). 7,7-Difluoro PGI₂analogs not only bind to IP strongly, but also bind to EP1-4 weakly(non-patent documents 4 and 5). However, a selective EP4 agonist, whichis one of the 7,7-difluoro PGI₂ analogs, shows weak binding affinity toIP, EP1, EP2 and EP3 and strongly and selectively binds only to EP4, hasnot been reported.

EP4 is expressed in the immune cells, inflammatory cells, digestiveorgans, blood vessels, neuronal cells, eyes, kidney, bone and the like,and EP4 agonists are researched and developed as a medicament of immunediseases, diseases of the digestive tract, cardiovascular diseases,cardiac diseases, neurological diseases, ophthalmic diseases, renaldiseases, hepatic diseases, bone diseases and the like.

The EP4 agonists inhibit TNF-α production, promote IL-10 production,suppress inflammation and immunoreaction, and are considered to beuseful for the prophylaxis and/or treatment of immune diseases orinflammatory diseases such as autoimmune diseases (e.g., amyotrophiclateral sclerosis, multiple sclerosis, Sjogren's syndrome, rheumatoidarthritis, systemic lupus erythematosus), post-transplantation rejectionand the like, asthma, neuronal cell death, arthritis, lung injury,pulmonary fibrosis, emphysema, bronchitis, chronic obstructive pulmonarydisease, hepatopathy, acute hepatitis, nephritis (acute nephritis,chronic nephritis), renal failure, systemic inflammation responsesyndrome, sepsis, hemophagocytic syndrome, macrophage activationsyndrome, Still's disease, Kawasaki disease, burn, systemic granuloma,ulcerative colitis, Crohn's disease, hypercytokinemia at dialysis,multiple organ failure, shock and psoriasis.

EP4 agonists are considered to be useful for the prophylaxis and/ortreatment of arteriosclerosis since they suppress activation ofmacrophages (non-patent document 6).

EP4 agonists are considered to be useful as an agent for the prophylaxisand/or treatment of angina pectoris or myocardial infarction, since theyhave a protective action against cardiac ischemia-reperfusion injury(non-patent document 7).

EP4 agonists are considered to be also useful as an agent for theprophylaxis and/or treatment of a brain disorder induced by cerebralhemorrhage, cerebral infarction, subarachnoid hemorrhage and the like,since they have a protective action against ischemia-reperfusion injuryin the brain as well (non-patent document 8).

EP4 agonists are considered to be also useful as an agent for theprophylaxis and/or treatment of an ischemia-reperfusion injury in theliver (non-patent document 9).

EP4 agonists are considered to be useful as an agent for the prophylaxisand/or treatment of glaucoma, since they have an intraocularpressure-lowering action (non-patent document 10).

EP4 agonists are considered to be also useful for the prophylaxis and/ortreatment of glomerulonephritis and diabetic nephritis, since EP4 isabundantly expressed in the renal glomerulus (non-patent document 11).

EP4 agonists are considered to be also useful for the prophylaxis and/ortreatment of calvities, alopecia and the like, since EP4 is alsoinvolved in hair growth and hair restoration (non-patent document 12).

EP4 agonists are considered to be useful as a (promoting) agent ofcervical ripening, since EP4 is also involved in cervical ripening(non-patent document 13).

EP4 agonists are considered to be useful as an agent for the prophylaxisand/or treatment of osteoporosis, or as a healing promoter of bonefracture, since EP4 is also involved in an osteogenic action (non-patentdocuments 14 and 15).

Since EP4 is expressed in blood vessels and EP4 agonists relax bloodvessels and contribute to increased blood flow, it is considered to beuseful for the prophylaxis and/or treatment of pulmonary arterialhypertension, peripheral arterial obstruction (arteriosclerosisobliterans and thromboangiitis obliterans) and various symptoms(intermittent claudication with lumbar spinal stenosis, leg numbness,Raynaud's syndrome, erectile dysfunction, hemorrhoids etc.) attributedto peripheral circulatory disturbance (non-patent documents 16-20).

EP4 is expressed in fibroblasts, and an EP4 agonist is considered topromote expression of basic fibroblast growth factor and is useful forpromotion of healing of pressure ulcer and wound (non-patent document21).

It has been reported that EP4 is expressed in the cochlea, and an EP4agonist is also useful for the prophylaxis and/or treatment of hearingdisorder caused by sound (non-patent document 22).

Inflammation of the digestive tract is observed in the mouth cavity,esophagus, stomach, small intestine, large intestine and anus, andincludes acute inflammation and chronic inflammation. When the mucosalepithelia are affected by physical or chemical stimuli, or are infectedby bacteria or virus, inflammation is induced, and erosions or ulcerouslesions occur depending on the level of the inflammation. Excessivesecretion of gastric acid due to a stress causes gastritis, gastriculcer or duodenal ulcer. In addition, excessive ingestion of alcoholinduces congestion of mucosal blood flow or reflux of gastric acid dueto reduced stomach motility, thus causing gastritis, gastric ulcer,duodenal ulcer or esophagitis. Orthopedic patients, rheumatoid arthritispatients and the like under a long term administration of anon-steroidal anti-inflammatory drug suffer from drug-induced gastriculcer or duodenal ulcer. In addition, cancer patients develop radiationenteritis with radiation therapy or drug-induced enteritis withanti-cancer drug treatment. Furthermore, patients infected withtuberculosis, amebic dysentery and the like develop infectiousenterogastritis such as intestinal tuberculosis and amebic colitis.Besides these, ischemic enteritis and the like are developed by ischemiadue to blood flow obstruction. If immunity of patients with inflammationof digestive tract is abnormal, even when the cause is removed, repairof the organ is prevented and conditions become chronic. Of theseinflammatory diseases of the digestive tract, the diseases withinflammation in the intestine are referred to as inflammatory boweldisease in a broad sense.

On the other hand, there are inflammatory intestinal diseases ofunidentified cause. Ulcerative colitis and Crohn's disease are two wellknown diseases, which are inflammatory bowel disease in a narrow sense.Furthermore, it also includes similar diseases such as intestinalBehcet's disease and simple ulcer. They are intractable chronicgastrointestinal diseases along with repeated remission and relapse,where main etiology of the disease is considered to be less protectionof the intestinal epithelium, or abnormal intestinal immune responseagainst enteric bacteria entering into the intestinal tissues.

Ulcerative colitis is a chronic colon disease in which erosions andulcers are formed in the large intestinal mucosa continuously from therectum, and symptoms thereof include abdominal pain, diarrhea, bloodystool, fever and the like. On the other hand, in Crohn's disease, alesion can occur in any digestive tract from the mouth cavity to largeintestine and anus. This disease is characterized by discontinuouslongitudinal ulcer and cobblestone-like appearance in thegastrointestinal tract, and the symptoms thereof include abdominal pain,diarrhea, fever, undernutrition due to malabsorption of nutrients,anemia, and the like.

For the prophylaxis and/or treatment of inflammation in inflammatorydiseases of the digestive tract, in case of with a known cause, thecause is removed or suppressed. For example, antacid, anticholinergicagent, histamine H2 receptor antagonist, proton pump inhibitor and thelike are used against inflammation in gastritis, gastric ulcer, duodenalulcer and the like to suppress secretion and actions of gastric acid. Inother instances, PGE derivatives and the like are used to supplementPGE₂ for inflammation induced by a non-steroidal anti-inflammatory drug,which inhibits PGE₂ production. However, PGI₂ derivatives are not used.

On the other hand, the prophylaxis or treatment of inflammatory boweldisease in a narrow sense includes drug therapy, nutrition (diet)therapy and surgical therapy. For the drug therapy, 5-aminosalicylicacid preparations (pentasa, salazopyrin), steroids (prednisolone),immunosuppressants (azathiopurine, mercaptopurine and tacrolimus),anti-TNF-α antibodies (infliximab) and the like are used. It has beenrecently reported that an EP4 agonist is effective for inflammatorybowel disease (non-patent documents 23-25).

In addition, since EP4 is also involved in mucosal-protective action, anEP4 agonist is considered to be useful for the prophylaxis and/ortreatment of gastrointestinal tract injury such as gastric ulcer,duodenal ulcer and the like, and stomatitis (non-patent document 26).

PRIOR ART DOCUMENTS Patent Documents

-   patent document 1: DE 2405255-   patent document 2: WO 03/103664-   patent document 3: WO 00/24727-   patent document 4: U.S. Pat. No. 7,402,605-   patent document 5: JP-A-7-330752-   patent document 6: JP-A-2004-256547

Non-Patent Documents

-   non-patent document 1: Biochim. Biophys. Acta, 1483: 285-293 (2000).-   non-patent document 2: Br. J. Pharmacol., 122: 217-224 (1997).-   non-patent document 3: J. Med. Chem., 22: 1340-1346 (1979).-   non-patent document 4: Prostaglandins, 53: 83-90 (1997).-   non-patent document 5: Br. J. Pharmacol., 134: 313-324 (2001).-   non-patent document 6: J. Biol. Chem., 283: 9692-9703 (2008).-   non-patent document 7: Cardiovasc. Res., 81: 123-132 (2009).-   non-patent document 8: Neurosci. Lett., 438: 210-215 (2008).-   non-patent document 9: Transplant. Proc., 37: 422-424 (2005).-   non-patent document 10: Exp. Eye Res., 89: 608-617 (2009).-   non-patent document 11: Kidney Int., 70: 1099-1106 (2006).-   non-patent document 12: Biochem. Biophys. Res. Commun., 290: 696-700    (2002).-   non-patent document 13: Biol. Reprod., 75: 297-305 (2006).-   non-patent document 14: Proc. Natl. Acad. Sci. USA., 99: 4580-4585    (2002).-   non-patent document 15: Expert Opin. Investig Drugs., 18: 746-766    (2009).-   non-patent document 16: Hypertension, 50: 525-530 (2007).-   non-patent document 17: Br. J. Pharmacol., 154: 1631-1639 (2008)-   non-patent document 18: Am. J. Respir. Crit. Care Med., 178: 188-196    (2008).-   non-patent document 19: Spine, 31: 869-872 (2006).-   non-patent document 20: Br. J. Pharmacol., 136: 23-30 (2002)-   non-patent document 21: Kobe J. Med. Sci., 47: 35-45 (2001).-   non-patent document 22: Neuroscience, 160: 813-819 (2009).-   non-patent document 23: J. Clin Invest., 109: 883-893 (2002).-   non-patent document 24: Scand. J. Immunol., 56: 66-75 (2002).-   non-patent document 25: J. Pharmacol. Exp. Ther., 320: 22-28 (2007).-   non-patent document 26: World J. Gastroenterol., 15: 5149-5156    (2009).

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention aims to provide a compound which is a novelprostaglandin I₂ derivative, superior in metabolic stability, andselectively binds to a specific prostaglandin receptor.

Means of Solving the Problems

In an attempt to solve the aforementioned problems, the presentinventors have synthesized novel PG analogs conferred with particularproperties of fluorine atom and conducted studies to clarify theproperty and physiological activity thereof. As a result, the inventorshave found that a novel 7,7-difluoro PGI₂ derivative, wherein thecarboxy group at C-1 of the prostanoic acid skeleton is substituted by atetrazole group and two fluorine atoms are bonded, is excellent in theproperty and pharmacological action, that unexpectedly, even though itis a PGI₂ derivative, it has a selective EP4 agonist activity andconsiderably loses an IP agonist activity, which is observed in thecarboxylate form at C-1, and that it is an excellent chemical as amedicament due to such agonist actions, which resulted in the completionof the present invention. The selective EP4 agonist can be an activeingredient of a medicament with reduced side effects via otherreceptors.

As far as the present inventors know, synthetic examples, property,physiological activity and the like of PGI₂ analogs, wherein the C-1 ofPG is a tetrazole group and two fluorine atoms are present at the C-7 ofPG, have not been published at all.

Therefore, the present invention provides a 7,7-difluoro PGI₂ derivativerepresented by the following formula (1), which is a selective EP4agonist (hereinafter sometimes to be abbreviated as compound (1) of thepresent invention), a pharmaceutically acceptable salt thereof, and amedicament containing the same as an active ingredient, and relates tothe following.

[1] A compound represented by the formula (1):

wherein R¹ and R² are each independently a hydrogen atom or a straightchain alkyl group having a carbon number of 1 to 3, and R³ is a hydrogenatom, an alkyl group having a carbon number of 1 to 4, an alkoxyalkylgroup, an aryl group, a halogen atom or a haloalkyl group), or apharmaceutically acceptable salt thereof.[2] The compound of [1], wherein R¹ is a methyl group, or apharmaceutically acceptable salt thereof.[3] The compound of [1] or [2], wherein R³ is a methyl group, or apharmaceutically acceptable salt thereof.[4] The compound of any of [1] to [3], wherein R² is a hydrogen atom, ora pharmaceutically acceptable salt thereof.[5] The compound of any of [1] to [4], wherein R¹ is a methyl group, andR² is a hydrogen atom, or a pharmaceutically acceptable salt thereof.[6] The compound of any of [1]-[5], wherein R³ is an m-methyl group, ora pharmaceutically acceptable salt thereof.[7] The compound of [1], wherein R¹ is a methyl group, R² is a hydrogenatom, and R³ is a methyl group, or a pharmaceutically acceptable saltthereof.[8] The compound of [1], wherein R¹ is a hydrogen atom, R² is a methylgroup, and R³ is a methyl group, or a pharmaceutically acceptable saltthereof.[9]4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane,or a pharmaceutically acceptable salt thereof.[10]4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane,or a pharmaceutically acceptable salt thereof.[11]4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4S)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane,or a pharmaceutically acceptable salt thereof.[12] A medicament comprising the compound of any of [1] to [11], or apharmaceutically acceptable salt thereof as an active ingredient.[13] A medicament for the prophylaxis or treatment of a disease of thedigestive tract, comprising the compound of any of [1] to [11], or apharmaceutically acceptable salt thereof as an active ingredient.[14] The medicament of [13], wherein the disease of the digestive tractis an inflammatory disease or ulcerative disease of the digestive tract.[15] The medicament of [14], wherein the inflammatory disease of thedigestive tract is an inflammatory bowel disease.[16] The medicament of [15], wherein the inflammatory bowel disease isulcerative colitis or Crohn's disease.[17] The medicament of [15], wherein the inflammatory bowel disease isintestinal Behcet's disease or simple ulcer.[18] The medicament of [14], wherein the ulcerative disease of thedigestive tract is esophagitis, esophageal ulcer, gastritis or gastriculcer.[19] The medicament of [18], wherein the gastritis or gastric ulcer isdrug-induced gastritis or gastric ulcer.[20] The medicament of [19], wherein the drug-induced gastritis orgastric ulcer is induced by a non-steroidal anti-inflammatory drug.[21] The medicament of [18], wherein the gastritis or gastric ulcer isinduced by alcohol.[22] The medicament of [14], wherein the ulcerative disease of thedigestive tract is small intestinal ulcer.[23] The medicament of [22], wherein the small intestinal ulcer isdrug-induced small intestinal ulcer.[24] The medicament of [23], wherein the drug-induced small intestinalulcer is induced by a non-steroidal anti-inflammatory drug.[25] The medicament of [22], wherein the small intestinal ulcer isinduced by alcohol.[26] An EP4 agonist comprising the compound of any of [1] to [11], or apharmaceutically acceptable salt thereof.[27] A medicament comprising the EP4 agonist of [26] as an activeingredient.[28] The medicament of [27] for the prophylaxis or treatment of adisease involving EP4.[29] The medicament of [28] for the prophylaxis or treatment of adisease whose symptoms can be mitigated by a selective EP4 agonistaction.[30] The medicament of [29], wherein the disease whose symptoms can bemitigated by a selective EP4 agonist action is an immune disease, acardiovascular disease, a cardiac disease, a respiratory disease, anophthalmic disease, a renal disease, a hepatic disease, a bone disease,a disease of the digestive tract, a neurological disease or a skindisease.[31] The medicament of [30], wherein the immune disease is amyotrophiclateral sclerosis, multiple sclerosis, Sjogren's syndrome, rheumatoidarthritis, systemic lupus erythematosus, post-transplantation rejection,arthritis, systemic inflammation response syndrome, sepsis,hemophagocytic syndrome, macrophage activation syndrome, Still'sdisease, Kawasaki disease, hypercytokinemia at dialysis, multiple organfailure, shock or psoriasis.[32] The medicament of [30], wherein the cardiovascular disease orcardiac disease is arteriosclerosis, angina pectoris, myocardialinfarction, brain disorder caused by cerebral hemorrhage, brain disordercaused by cerebral infarction, brain disorder caused by subarachnoidhemorrhage, pulmonary arterial hypertension, peripheral arterialobstruction (arteriosclerosis obliterans and thromboangiitis obliterans)or various symptoms attributed to peripheral circulatory disturbance(intermittent claudication or leg numbness caused by lumbar spinalstenosis, Raynaud's syndrome, erectile dysfunction, hemorrhoids etc.).[33] The medicament of [30], wherein the respiratory disease is asthma,lung injury, pulmonary fibrosis, emphysema, bronchitis or chronicobstructive pulmonary disease.[34] The medicament of [30], wherein the ophthalmic disease is glaucomaor ocular hypertension.[35] The medicament of [30], wherein the renal disease isglomerulonephritis, diabetic nephropathy, IgA nephropathy or renalischemia-reperfusion injury.[36] The medicament of [30], wherein the hepatic disease is hepatitis,hepatopathy or hepatic ischemia-reperfusion injury.[37] The medicament of [30], wherein the bone disease is osteoporosis,bone fracture or a postoperative recovery phase after osteotomy.[38] The medicament of [30], wherein the neurological disease isneuronal cell death.[39] The medicament of [30], wherein the skin disease is pressure ulceror wound.[40] The medicament of [28], wherein the disease involving EP4 is adisease selected from the group consisting of calvities, alopecia,cervical ripening failure and a hearing disorder.

Effect of the Invention

The novel 7,7-difluoro PGI₂ derivative afforded by the present inventioncan provide a medicament which maintains blood concentration for a longtime and exhibits a pharmacological action by parenteral administrationor oral administration, and which is for the prophylaxis or treatment ofinflammation of the digestive tract or the onset of diarrhea or bloodfeces in inflammatory bowel disease, or for the prophylaxis or treatmentof gastritis or ulcer in gastric ulcer, small intestinal ulcer and thelike. Furthermore, due to the EP4 agonist action, a medicament for theprophylaxis or treatment of immune diseases, cardiovascular diseases,cardiac diseases, respiratory diseases, ophthalmic diseases, renaldiseases, hepatic diseases, bone diseases, diseases of the digestivetract, neurological diseases, skin diseases and the like can beprovided. In clinical situations, the compound of the present inventionis expected to show similar efficacy in the disease group for which anEP4 agonist can provide effects, whereas the concern of side effectssuch as hemorrhage, hypotension, palpitation and face flush is lowerbecause of its weaken IP agonist action on the circulatory system.Particularly, the compound of the present invention is effective, basedon the EP4 agonist action, for inflammation of the digestive tractassociated with immunity, drug-induced mucosal injury of the digestivetract, injury of the digestive tract and delayed healing due to mucosalregenerative disorder, ophthalmic diseases, renal diseases, and hepaticdiseases. Specifically, the compound is useful for inflammatory boweldisease such as ulcerative colitis and Crohn's disease, alcoholicgastritis, gastric ulcer and small intestinal ulcer, nephritis,glaucoma, ocular hypertension, hepatitis and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the effect on blood pressure in mice.

FIG. 1B shows the effect on heart rate in mice.

FIG. 2A shows the effect on abnormal stool in mouse (BALB/c) DSS colitismodel.

FIG. 2B shows the effect on colon shortening in mouse (BALB/c) DSScolitis model.

FIG. 3A shows the effect of BPS on abnormal stool in mouse (C57BL/6) DSScolitis model.

FIG. 3B shows the effect on abnormal stool in mouse (C57BL/6) DSScolitis model.

FIG. 3C shows the effect of BPS on colon shortening in mouse (C57BL/6)DSS colitis model.

FIG. 3D shows the effect on colon shortening in mouse (C57BL/6) DSScolitis model.

FIG. 4A shows the effect on abnormal stool in rat DSS colitis model.

FIG. 4B shows the effect on colon shortening in rat DSS colitis model.

FIG. 4C shows the effect on colonic tissue injury in rat DSS colitismodel.

FIG. 5 shows the effect on abnormal stool in remission/relapse model ofmouse DSS colitis.

FIG. 6A shows the effect on stool consistency score in mouse T celltransfer model of colitis model.

FIG. 6B shows the effect on fecal occult blood score in mouse T celltransfer model of colitis.

FIG. 6C shows the effect on body weight decrease score in mouse T celltransfer model of colitis.

FIG. 6D shows the effect on DAI score in mouse T cell transfer model ofcolitis.

FIG. 7 shows the effect on gastric ulcer in rat ethanol induced-gastricmucosal injury model.

FIG. 8 shows the effect on small intestinal ulcer in ratindomethacin-induced small intestinal injury model.

FIG. 9A shows the effect on urine volume in rat anti Thy-1antibody-induced glomerulonephritis model.

FIG. 9B shows the effect on the amount of urine protein in rat antiThy-1 antibody induced glomerulonephritis model.

FIG. 9C shows the effect on relative kidney weight in rat anti Thy-1antibody-induced glomerulonephritis model.

FIG. 9D shows the effect on renal histopathology (total glomerular cellcount) in rat anti Thy-1 antibody-induced glomerulonephritis model.

FIG. 9E shows the effect on renal histopathology (mesangial region) inrat anti Thy-1 antibody-induced glomerulonephritis model.

FIG. 9F shows the effect on renal histopathology (PCNA-positiveglomerular cell count) in rat anti Thy-1 antibody-inducedglomerulonephritis model.

FIG. 10 shows the effect on rabbit intraocular pressure.

FIG. 11 shows the prophylactic effect in mouse concanavalin A-inducedhepatitis model.

EMBODIMENT OF THE INVENTION Definition

In the present specification, the “selective EP4 agonist” means acompound which shows a weak agonist action (pharmacological activity) onPGI₂ receptor (IP) relative to the agonist action generally found inPGI₂ analogs, and has a remarkably superior agonist action on PGE₂receptor subtype EP4 as compared to IP agonist action. The EP4 agonistaction can be measured in accordance with the measurement method of theagonist activity described in the below-mentioned Example 19. The IPagonist action can be measured in accordance with the method describedin Example 20. Whether a compound is a selective EP4 agonist can beevaluated by measuring the ratio of binding inhibition constant Kivalues of EP4 and IP (IP/EP4 ratio) in the same species in accordancewith the measurement method described in Example 18. Examples of theselective EP4 agonist include a compound having the aforementioned ratioof not less than 5, preferably not less than 10, more preferably notless than 50, most preferably not less than 100.

In the present specification, the “prostaglandin I₂ derivative” means acompound with a structure modified by a general technique in the organicchemistry, based on the structure of natural type PGI₂. In thefollowing, the compound of the present invention is explained.

(Definition of the Compound of the Present Invention)

In the nomenclature of the compounds in the present specification, thenumbers used to show the position in PG skeleton correspond to thenumbers in the prostanoic acid skeleton. In the present specification, agroup in which a hydrogen atom of an alkyl group is substituted is alsoindicated as a substituted alkyl group. The same applies to othergroups.

In addition, a “lower” organic group such as alkyl group and the likemeans that the carbon number thereof is 1 to 6. The carbon number of the“lower” organic group is preferably 1 to 4.

The “alkyl group” may be a straight chain or a branched chain. Unlessotherwise specified, the alkyl group is preferably a lower alkyl grouphaving a carbon number of 1 to 6, and a lower alkyl group having acarbon number of 1 to 4 is particularly preferable. Examples of thealkyl group include a methyl group, an ethyl group, a propyl group, anisopropyl group, a butyl group, an isobutyl group, a sec-butyl group, atert-butyl group, a pentyl group, a hexyl group and the like.

The “alkoxy group” is preferably a lower alkoxy group having a carbonnumber of 1 to 6, particularly preferably an alkoxy group having acarbon number of 1 to 4. The alkoxy group may be a straight chain or abranched chain. Examples of the alkoxy group include a methoxy group, anethoxy group, a propoxy group, a butoxy group and the like.

The “alkoxyalkyl group” is an alkyl group substituted by an alkoxygroup. The alkoxy group of the alkoxyalkyl group is preferably a loweralkoxy group having a carbon number of 1 to 4, and the alkyl group ofthe alkoxyalkyl group is preferably a lower alkyl group having a carbonnumber of 1 to 4. The alkoxyalkyl group is preferably a loweralkoxyalkyl group (that is, the carbon number of the whole alkoxyalkylgroup is 1 to 6), more preferably a lower alkoxyalkyl group having acarbon number of 1 to 4. Examples of the alkoxyalkyl group include amethoxymethyl group, an ethoxymethyl group, a propoxymethyl group, anethoxyethyl group and the like.

The “aryl group” is a monovalent aromatic hydrocarbon group optionallyhaving substituent(s). As an aryl group without a substituent, a phenylgroup is preferable.

As the “substituted aryl group” (an aryl group having substituent(s)),an aryl group wherein one or more hydrogen atoms in the aryl group aresubstituted by a lower alkyl group, a halogen atom, a halogenated (loweralkyl) group, a lower alkoxy group and the like is preferable.Preferable examples of the substituted aryl group include a substitutedphenyl group, and particular examples thereof include a monohalophenylgroup (e.g., chlorophenyl group, fluorophenyl group, bromophenyl groupetc.), a (halogenated lower alkyl) substituted phenyl group (e.g.,trifluoromethylphenyl group etc.) and a (lower alkoxy) phenyl group(e.g., methoxyphenyl group, ethoxyphenyl group etc.).

The “halogen atom” is a fluorine atom, a chlorine atom, a bromine atomor an iodine atom.

The “haloalkyl group” is an alkyl group wherein one or more hydrogenatoms in the alkyl group are substituted by a halogen atom, andpreferred is a lower haloalkyl group having a carbon number of 1 to 6.Examples of the haloalkyl group include a fluoromethyl group, adifluoromethyl group, a trifluoromethyl group, a trifluoroethyl group, apentafluoroethyl group, a chloromethyl group, a bromomethyl group andthe like.

As compound (1) of the present invention, the following compound ispreferable from the aspects of pharmacological activity and physicalproperty.

That is, R¹ and R² are each independently a hydrogen atom or a straightchain alkyl group having a carbon number of 1 to 3, and eachindependently is preferably a hydrogen atom or a methyl group.Particularly preferably, one of R¹ and R² is a hydrogen atom, and theother is a methyl group.

R³ is a hydrogen atom, an alkyl group having a carbon number of 1 to 4,an alkoxyalkyl group, an aryl group, a halogen atom or a haloalkylgroup, and a hydrogen atom, an alkyl group having a carbon number of 1to 4, a lower alkoxyalkyl group such as a methoxymethyl group and thelike, a halogen atom such as a chlorine atom, a fluorine atom and thelike, or a lower haloalkyl group such as a lower fluoroalkyl group andthe like is preferable. Particularly, a hydrogen atom, an alkyl grouphaving a carbon number of 1 to 4, a chlorine atom or a haloalkyl havinga carbon number of 1 to 4 is preferable. As the alkyl group having acarbon number of 1 to 4, a methyl group and an ethyl group arepreferable, and as the haloalkyl group having a carbon number of 1 to 4,a trifluoromethyl group is preferable.

As R³, a hydrogen atom, a methyl group or a trifluoromethyl group ismost preferable.

In addition, R³ may be substituted at any of the ortho(o), meta(m) andpara(p) positions relative to the position of substitution of the mainchain of the prostaglandin skeleton by a benzene ring. R³ isparticularly preferably substituted at the meta(m) position.

(Embodiment of Preferable Compound of the Present Invention)

In addition, preferable combinations of R¹, R² and R³ in compound (1) ofthe present invention are as follows.

R¹ is a hydrogen atom, R² is a hydrogen atom, and R³ is a hydrogen atom.

R¹ is a hydrogen atom, R² is a hydrogen atom, and R³ is a methyl group.

R¹ is a hydrogen atom, R² is a hydrogen atom, and R³ is a chlorine atom.

R¹ is a hydrogen atom, R² is a hydrogen atom, and R³ is atrifluoromethyl group.

R¹ is a methyl group, R² is a hydrogen atom, and R³ is a hydrogen atom.

R¹ is a methyl group, R² is a hydrogen atom, and R³ is a methyl group.

R¹ is a methyl group, R² is a hydrogen atom, and R³ is a chlorine atom.

R¹ is a methyl group, R² is a hydrogen atom, and R³ is a trifluoromethylgroup.

R¹ is a hydrogen atom, R² is a methyl group, and R³ is a hydrogen atom.

R¹ is a hydrogen atom, R² is a methyl group, and R³ is a methyl group.

R¹ is a hydrogen atom, R² is a methyl group, and R³ is a chlorine atom.

R¹ is a hydrogen atom, R² is a methyl group, and R³ is a trifluoromethylgroup.

R¹ is a methyl group, R² is a methyl group, and R³ is a hydrogen atom.

R¹ is a methyl group, R² is a methyl group, and R³ is a methyl group.

R¹ is a methyl group, R² is a methyl group, and R³ is a chlorine atom.

R¹ is a methyl group, R² is a methyl group, and R³ is a trifluoromethylgroup.

Furthermore, preferable combinations from among those mentioned aboveare as follows, since the selective EP4 agonist action is high.

R¹ is a methyl group, R² is a hydrogen atom, and R³ is a methyl group.

R¹ is a hydrogen atom, R² is a methyl group, and R³ is a methyl group.Moreover, most preferable combinations are as follows.

R¹ is a methyl group, R² is a hydrogen atom, and R³ is a m-methyl group.

R¹ is a hydrogen atom, R² is a methyl group, and R³ is a m-methyl group.

(Production Method of Compound (1) of the Present Invention)

Compound (1) of the present invention can be produced, for example,based on the methods described in JP-A-07-324081 and JP-A-08-217772relating to the inventions made by the present inventors. For example,using Corey lactone as a starting material, ω chain is introduced atfirst, and the lactone is converted by fluorination into ωchain-containing difluoro Corey lactone. Then, an α chain unit isintroduced by an addition reaction with an organometallic reagent havinga tetrazole group at the terminal and a dehydrating reaction, or Wittigreaction using a phosphonium salt having a tetrazole group at theterminal, and the like, and the hydroxyl group is deprotected asnecessary, whereby compound (1) can be synthesized.

Alternatively, difluoro Corey lactone is obtained by fluorination fromCorey lactone as a starting material. Then, an α chain unit isintroduced by an addition reaction with an organometallic reagent havinga tetrazole group at the terminal and a dehydrating reaction, or Wittigreaction using a phosphonium salt having a tetrazole group at theterminal, and the like, ω chain is introduced, and the hydroxyl group isdeprotected as necessary, whereby compound (1) can be synthesized.

Alternatively, compound (1) can also be synthesized by converting acarboxy group of the carboxylic acid derivative described inJP-A-07-324081 to a cyano group and converting the derivative to atetrazole derivative.

Of these production methods, representative methods are specificallyexplained using the following chemical formulas.

For example, using Corey lactone (7) as a starting material, ω chain isintroduced at first, the obtained Corey lactone derivative (6)containing the ω chain is subjected to a fluorination reaction to give ωchain containing difluoro Corey lactone derivative (3) having twofluorine atoms at the α-position of the carbonyl group. Then, thedifluorolactone derivative (3) is reacted with phosphorane derivative(4) to introduce an α chain unit, whereby PGI₂ derivative (2) withprotected hydroxyl groups can be obtained. The hydroxyl-protecting groupis removed to give compound (1) of the present invention.

The phosphorane derivative (4) can be obtained from a phosphonium saltderivative (5).

Except when R¹-R³ are particular substituents, the above-mentionedlactone derivative (6) is a known compound. The above-mentioned novellactone derivative (6) wherein R¹-R³ are particular substituents can beproduced by a method similar to that of known lactone derivatives (6).For example, novel lactone derivatives (6) can be produced by reacting3-aryl-2-oxoalkylphosphonic acid diester with Corey lactone having aformyl group. Here, the alkyl chain of alkylphosphonic acid has a carbonnumber of not less than 3.

R⁴, R⁵ and R⁷ are each independently a hydroxyl-protecting group. R⁴,R⁵, and R⁷ may be same protecting groups. As the protecting group, thehydroxyl-protecting group described in “Shin Jikken Kagaku Koza (NewCourses in Experimental Chemistry) 14, synthesis and reaction of organiccompound (V)” (Maruzen Company, Limited), “Protective Groups in Organicsynthesis” (by T. W. Greene, J. Wiley & Sons) and the like can be used.Specifically, a triorganosilyl group, an alkoxyalkyl group, a monovalentgroup having a cyclic ether structure and the like can be mentioned. Asthe triorganosilyl group, a silyl group wherein 3 groups selected froman alkyl group, an aryl group, an aralkyl group and alkoxy group arebonded to a silicon atom is preferable, and a group wherein 3 loweralkyl groups or aryl groups are bonded to a silicon atom is particularlypreferable. As specific examples of the protecting group, atetrahydropyranyl group, a tert-butyldimethylsilyl group, atert-butyldiphenylsilyl group, a triethylsilyl group, a triphenylsilylgroup or a triisopropylsilyl group and the like are preferable.Particularly, a tetrahydropyranyl group, a tert-butyldimethylsilylgroup, a tert-butyldiphenylsilyl group and the like are preferable.

The hydroxyl-protecting group can be removed easily. The deprotectionmethod of the protected hydroxyl group can be a conventional method. Forexample, the methods described in “Shin Jikken Kagaku Koza (New Coursesin Experimental Chemistry) 14 synthesis and reaction of organic compound(I), (II) and (V)” (Maruzen Company, Limited), “Protective Groups inOrganic synthesis” (by T. W. Greene, J. Wiley & Sons) and the like canbe employed.

For conversion of lactone derivative (6) to difluorolactone derivative(3) by a fluorination reaction, various known fluorination methods canbe applied. For example, a method including reacting with variouselectrophilic fluorinating agents in an inert solvent can be employed.The fluorination can also be performed by the methods described inJP-A-07-324081 and JP-A-09-110729 relating to the invention by thepresent inventors.

In the fluorination reaction of lactone derivative (6), an electrophilicfluorinating agent is preferably used. As the electrophilic fluorinatingagent, known or well known electrophilic fluorinating agent can be used.For example, the electrophilic fluorinating agents described in“Chemistry of fluorine” (Kodansha Scientifics Ltd.) by Tomoya Kitazume,Takashi Ishihara, and Takeo Taguchi and the like can be mentioned.Specifically, N-fluorosulfonyl amides, N-sulfonyl imide derivative,acetyl hypofluorite, fluorine gas and the like can be mentioned.

The electrophilic fluorinating agent is preferably used in the presenceof an inert solvent. As the inert solvent, ether solvents, hydrocarbonsolvents, polar solvents, mixed solvents thereof and the like can bementioned.

The difluorolactone derivative (3) obtained by the fluorination reactionis then reacted with phosphorane derivative (4) to give PGI₂ derivative(2) wherein the hydroxyl group is protected. The phosphorane derivative(4) is produced from the corresponding phosphonium salt derivative (5),in an inert solvent in the presence of a base, and the formedphosphorane derivative (4) is preferably used directly for the Wittigreaction with difluorolactone derivative (3) without isolation. As theproduction methods of phosphorane derivative (4) and phosphonium saltderivative (5), the methods described in DE2242239, DE2405255 and thelike can be employed. As R⁶ for phosphorane derivative (4) orphosphonium salt derivative (5), an aryl group such as a phenyl group, atolyl group and the like is preferable, and a phenyl group isparticularly preferable. As the inert solvent, ether solvents,hydrocarbon solvents, polar solvents, aqueous solvents, alcoholicsolvents, mixed solvents thereof and the like can be mentioned.

The hydroxyl-protecting group is removed from the PGI₂ derivative (2)obtained by the above method to give compound (1).

Since compound (1) of the present invention has an asymmetric carbon inthe structure, various stereoisomers and optical isomers are present.The present invention encompasses all of such stereoisomers, opticalisomers, and mixtures thereof.

Specific examples of compound (1) of the present invention include thecompound represented by the following formula (8).

(Examples of Compound (1) of the Present Invention)

A compound wherein, in the formula (8), R¹, R², and R³ have structuresshown in the following Table 1 can be mentioned.

TABLE 1 R¹ R² R³ Compound A H H H Compound B H H Me Compound C H H ClCompound D H H CF₃ Compound E Me H H Compound F Me H Me Compound G Me HCl Compound H Me H CF₃ Compound I H Me H Compound J H Me Me Compound K HMe Cl Compound L H Me CF₃ Compound M Me Me H Compound N Me Me MeCompound O Me Me Cl Compound P Me Me CF₃

(Features of Compound (1) of the Present Invention)

Compound (1) of the present invention is a PGI₂ derivative which is noteasily metabolized in the body and has improved stability. Since thecarboxy group of the PG skeleton is converted to a tetrazole group, itis not easily metabolized by β-oxidation, which is known as a commonmetabolic pathway of fatty acid such as prostaglandins. Therefore, ithas a prolonged plasma half-life and can maintain an effective plasmaconcentration for a long time, as compared to a compound having acarboxy group of the PG skeleton. Since the metabolic stability isimproved in this way, the bioavailability of drugs can be improved.

Compound (1) of the present invention or a pharmaceutically acceptablesalt thereof shows an action of a selective EP4 agonist. Examples ofpreferable compound (1) showing such action are the same as theaforementioned preferable examples of compound (1).

(Medicament Containing Compound (1) of the Present Invention or aPharmaceutically Acceptable Salt Thereof as Active Ingredient)

The medicament of the present invention contains compound (1) and/or apharmaceutically acceptable salt of compound (1), and further, apharmaceutically acceptable carrier and, in some cases, other treatmentcomponents.

The medicament of the present invention contains compound (1) and/or apharmaceutically acceptable salt of compound (1), or a hydrate thereof,and further, a pharmaceutically acceptable carrier and, in some cases,other treatment components.

When the prophylactic or therapeutic agent of the present invention isadministered to patients, the daily dose varies depending on the age andbody weight of patients, pathology and severity and the like. Generally,0.0001-10 mg, preferably 0.01-1 mg, of the agent is desirablyadministered in one to several portions. For example, for oraladministration, 0.001-3 mg is preferable, and 0.001-0.5 mg isparticularly preferable. For intravenous administration, 0.0001-1 mg ispreferable, and 0.001-0.1 mg is particularly preferable. The dose can bechanged as appropriate depending on the disease and its condition. Asthe dosing regimen, an injection product of the agent may be desirablyadministered by continuous drip infusion.

For use as a medicament, the agent can be administered to the body byoral administration and parenteral administration (e.g., intravascular(intravenous, intraarterial) administration, subcutaneousadministration, rectal administration etc.). Examples of the dosage forminclude oral dosage form such as tablet, capsule and syrup, parenteraldosage form such as liquid injection (solution, emulsion, suspension andthe like), infusion, suppositories, nasal preparations, patches andinhalations. Oral dosage is particularly desirable.

A preparation in the aforementioned dosage form can be produced bymixing compound (1) of the present invention or a pharmaceuticallyacceptable salt thereof with additives necessary for formulation such asconventional carriers, excipients, binders and stabilizers, andformulating the mixture in a conventional method. For example, when thepreparation is a powder, granule, tablet and the like, it can beproduced by using any pharmaceutical carriers preferable for producing asolid dosage form, for example, excipients, lubricants, disintegrants,binders and the like.

These excipient may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate and sodiumphosphate; granulating agent and disintegrant, such as cornstarch andalginic acid; binder, such as starch, gelatin and gum arabic, andlubricant, such as magnesium stearate, stearic acid and talc. The tabletmay be uncoated or coated by a known technique to delay disintegrationand absorption in the stomach and the intestine, thus ensuring asustained release for a longer time. For example, a time delay material,such as glyceryl monostearate or glyceryl distearate may be used.

Compound (1) of the present invention may be provided as a hard gelatincapsule containing a mixture with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin. Alternatively, it may beprovided as a soft gelatin capsule containing a mixture with a watermiscible solvent, such as propylene glycol, polyethylene glycol andethanol, or oils, such as peanut oil, liquid paraffin and olive oil.

When the preparation is syrup or liquid, stabilizers, suspending agents,corrigents, aromatic substances and the like may be appropriatelyselected and used for the production, for example. For injectionmanufacturing, an active ingredient is dissolved in distilled water forinjection together with a pH adjuster such as hydrochloric acid, sodiumhydroxide, lactose, sodium lactate, acetic acid, disodium hydrogenphosphate and sodium dihydrogen phosphate, and an isotonic agent such assodium chloride and glucose, and injection is aseptically prepared. Aninactive nonaqueous diluent such as propylene glycol, polyethyleneglycol, olive oil, ethanol and polysorbate 80 may be used forformulation of the preparation. Moreover, mannitol, dextrin,cyclodextrin, gelatin and the like may be added, and the mixture isfreeze-dried in vacuo to give an injection to be dissolved before use.For stabilization and improvement of drug delivery to a lesion,moreover, a liposome preparation or a lipid emulsion may be formulatedby a known method and used as an injection.

In addition, a rectal dosage preparation may be produced by using asuppository base, such as cacao butter, fatty acid triglyceride, fattyacid diglyceride, fatty acid monoglyceride and polyethylene glycol.Furthermore, a water-soluble base, such as polyethylene glycol,polypropylene glycol, glycerol and glycerolgelatin, an oily base, suchas white petrolatum, hard fat, paraffin, liquid paraffin, Plastibase,lanolin and purified lanolin, and the like may be adjusted to suitableviscosity and ointment for intrarectal administration can also beproduced.

Compound (1) of the present invention or a pharmaceutically acceptablesalt thereof can be administered topically to the skin or mucousmembrane, i.e., transdermal or transmucosal administration. As generaldosage forms for this purpose, gel, hydrogel, lotion, solution, cream,ointment, sprays, dressing agent, foam preparation, film, skin patch,oblate, implant, sponge, fiber, bandage, microemulsion and the like canbe mentioned. As commonly-used carriers, alcohol, water, mineral oil,liquid paraffin, white petrolatum, glycerol, polyethylene glycol,propylene glycol and the like can be mentioned.

Compound (1) of the present invention can be mixed with cyclodextrin oran appropriate derivative thereof or a soluble polymer such aspolyethylene glycol-containing polymer, for the purpose of use in any ofthe aforementioned dosage forms, and improving solubility, dissolutionrate, bioavailability and stability. For example, drug-cyclodextrincomplex and the like have been confirmed to be generally useful for mostdosage forms and administration routes. Both inclusion and non-inclusioncomplexes can be used. As another method for direct complexation withdrugs, cyclodextrin can also be used as an auxiliary additive, i.e.,carrier, excipient or solubilizer. For these purposes, α-, β- andγ-cyclodextrins and the like are generally used.

(Pharmaceutically Acceptable Salt of Compound (1) of the PresentInvention)

A pharmaceutically acceptable salt of compound (1) of the presentinvention is a salt of the moiety of the tetrazole group of thederivative with a basic substance, which is a compound wherein thehydrogen atom of the tetrazole group is substituted by cation.

Examples of the cation include alkali metal cations such as Na⁺ and K⁺,metal cations (other than alkali metal cations) such as ½ Ca²⁺, ½ Mg²⁺,½ Zn²⁺ and ⅓ Al³⁺, NH₄+, ammonium cations of organic amine and aminoacid such as triethanolamine, diethanolamine, ethanolamine,tromethamine, lysine and arginine, and the like. Preferable cation issodium ion or potassium ion.

More particularly, the acceptable salt is a salt produced from apharmaceutically acceptable nontoxic base such as inorganic base andorganic base. As a salt derived from the pharmaceutically acceptablenontoxic inorganic base, lithium salt, copper salt, ferric salt, ferroussalt, manganic salt, manganese salt and the like can be mentioned inaddition to the aforementioned sodium salt, potassium salt, calciumsalt, magnesium salt, zinc salt, aluminum salt, ammonium salt and thelike. Of these, sodium salt, potassium salt, calcium salt, magnesiumsalt and ammonium salt are preferable, and sodium salt and potassiumsalt are particularly preferable. A salt derived from a pharmaceuticallyacceptable nontoxic organic base includes salts with primary, secondaryand tertiary amine, substituted amine including naturally occurringsubstituted amine, cyclic amine, and basic ion exchange resin. Otherthan the examples of the aforementioned organic amine and amino acid,isopropylamine, diethylamine, triethylamine, trimethylamine,tripropylamine, ethylenediamine, N,N′-dibenzylethylenediamine,2-diethylaminoethanol, 2-dimethylaminoethanol, morpholine,N-ethyl-morpholine, piperazine, piperidine, N-ethylpiperidine, betaine,caffeine, choline, glucamine, glucosamine, histidine, Hydrabamine,methyl glucamine, polyamine resin, procaine, purine, theobromine and thelike can be mentioned.

(Use of Medicament Containing Compound (1) of the Present Invention or aPharmaceutically Acceptable Salt Thereof as an Active Ingredient)

A medicament containing compound (1) of the present invention or apharmaceutically acceptable salt thereof as an active ingredient can beapplied to a disease involving EP4, preferably a disease wherein aselective EP4 agonist action can mitigate the symptom. Specifically, itis useful for immune diseases, diseases of the digestive tract,cardiovascular diseases, cardiac diseases, respiratory diseases,neurological diseases, ophthalmic diseases, renal diseases, hepaticdiseases, bone diseases, skin diseases and the like.

The immune disease in the present invention includes autoimmune diseasessuch as amyotrophic lateral sclerosis, multiple sclerosis, Sjogren'ssyndrome, rheumatoid arthritis and systemic lupus erythematosus,post-transplantation rejection and the like, and inflammatory diseasessuch as asthma, neuronal cell death, arthritis, lung injury, pulmonaryfibrosis, emphysema, bronchitis, chronic obstructive pulmonary disease,hepatopathy, acute hepatitis, nephritis (acute nephritis, chronicnephritis), renal failure, systemic inflammation response syndrome,sepsis, hemophagocytic syndrome, macrophage activation syndrome, Still'sdisease, Kawasaki disease, burn, systemic granuloma, hypercytokinemia atdialysis, multiple organ failure, shock and psoriasis.

The disease of the digestive tract in the present invention includesinflammatory disease and ulcerative disease of the digestive tract,which is a disease with inflammation or ulcer in the epithelial, mucosalor submucosal tissues of the digestive tract, or abnormal proliferationor dysfunction of mucosal epithelium, and which is caused by physicalstimuli, chemical stimuli such as by gastric juice, stimuli by drug suchas non-steroidal anti-inflammatory drugs and steroids, immune diseasesand autoimmune diseases of unknown etiology, mental diseases and thelike.

The inflammatory disease of the digestive tract includes inflammatorybowel disease, particularly ulcerative colitis, Crohn's disease, whichis a non-specific granulomatous inflammatory disease accompanied byfibrillization or ulceration, intestinal Behcet's disease and simpleulcer. The ulcerative disease of the digestive tract of the presentinvention includes stomatitis, aphthous stomatitis, esophagitis,esophageal ulcer, gastritis, gastric ulcer and small intestinal ulcer.

Moreover, gastritis and gastric ulcer include drug-induced gastritis,gastric ulcer, alcoholic gastritis and gastric ulcer, and thedrug-induced gastritis and gastric ulcer include gastritis and gastriculcer induced by a non-steroidal anti-inflammatory drug.

Small intestinal ulcer includes drug-induced small intestinal ulcer andalcoholic small intestinal ulcer, and the drug-induced small intestinalulcer includes small intestinal ulcer induced by a non-steroidalanti-inflammatory drug.

Particularly, the medicament of the present invention is useful as aprophylactic or therapeutic agent for ulcerative colitis, Crohn'sdisease, gastritis, gastric ulcer or small intestinal ulcer.

The cardiovascular disease and cardiac disease include arteriosclerosis,angina pectoris, myocardial infarction, brain disorder caused bycerebral hemorrhage, brain disorder caused by cerebral infarction, braindisorder caused by subarachnoid hemorrhage, pulmonary arterialhypertension, peripheral arterial obstruction (arteriosclerosisobliterans, and thromboangiitis obliterans) and various symptoms(intermittent claudication with lumbar spinal stenosis, leg numbness,Raynaud's syndrome, erectile dysfunction, hemorrhoids etc.) attributedto peripheral circulatory disturbance.

The respiratory disease includes asthma, lung injury, pulmonaryfibrosis, emphysema, bronchitis, and chronic obstructive pulmonarydisease.

The neurological disease includes neuronal cell death, amyotrophiclateral sclerosis, multiple sclerosis and brain disorder (braindisorders caused by cerebral hemorrhage, cerebral infarction, andsubarachnoid hemorrhage).

The ophthalmic disease includes glaucoma and ocular hypertension.

The renal disease includes glomerulonephritis, diabetic nephropathy, IgAnephropathy and renal ischemia-reperfusion injury.

The hepatic disease includes hepatitis, hepatopathy and hepaticischemia-reperfusion injury.

The bone disease includes osteoporosis, bone fracture, and apostoperative recovery phase after osteotomy.

The skin disease includes pressure ulcer and wound.

Furthermore, a medicament containing compound (1) of the presentinvention or a pharmaceutically acceptable salt thereof as an activeingredient is also useful as a prophylactic and/or therapeutic agent foralopecia, calvities, or hearing disorder (e.g., hearing disorder causedby sound), or a cervical ripening (promoting) agent.

The present invention is explained in detail in the following byreferring to specific examples, which are not to be construed aslimitative.

Example 1 Synthesis of methyl (2R)-2-(m-tolyl)propionate

To (2R)-2-(m-tolyl)propionic acid (12.45 g) were added methanol (14.83g) and concentrated sulfuric acid (6.46 g), and the mixture was stirredunder refluxing for 6 hr. Then, the mixture was neutralized with 10%aqueous sodium carbonate solution, and extracted with hexane. Afterdrying over magnesium sulfate, the residue was concentrated underreduced pressure to give the title compound (12.79 g). The structuralproperty was as described below.

¹H-NMR (CDCl₃): δ 1.49 (d, J=7.0 Hz, 3H), 2.33 (s, 3H), 3.64 (s, 3H),3.69 (dd, J=14.4, 7.3 Hz, 1H), 7.06-7.22 (m, 4H).

Example 2 Synthesis of dimethyl (3R)-2-oxo-3-(m-tolyl)butylphosphonate

To dimethyl methylphosphonate (1.97 g) was added tetrahydrofuran (THF)(25 mL), and the mixture was cooled to −78° C. n-Butyllithium (1.5 Mhexane solution) (10 mL) was added, and the mixture was stirred for 1hr. Then, a solution of methyl ester {methyl (2R)-2-(m-tolyl)propionate}synthesized in Example 1 (1.34 g) in THF (3.8 mL) was added at −78° C.,and the mixture was stirred for 2 hr. The reaction was quenched with 25mL of saturated aqueous sodium hydrogen carbonate, and the mixture wasextracted with ethyl acetate. The extract was dried over magnesiumsulfate, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (hexane/ethyl acetate5:1-1:5) to give the title compound (1.63 g). The structural propertywas as described below.

¹H-NMR (CDCl₃): δ 1.39 (d, J=6.7 Hz, 3H), 2.34 (s, 3H), 2.84 (ddd,J=22.3, 14.1, 0.6 Hz, 1H), 3.18 (dd, J=22.3, 14.1 Hz, 1H), 3.76 (dd,J=19.3, 11.1 Hz, 6H), 4.00 (dd, J=13.8, 7.0 Hz, 1H), 7.01-7.24 (m, 4H).

Example 3 Synthesis of(1S,5R,6R,7R)-6-[(1E,4R)-3-oxo-4-(m-tolyl)-1-pentenyl]-7-benzoyloxy-2-oxabicyclo[3.3.0]octan-3-one

Sodium hydride (55%) (8.75 g) was dispersed in 1,2-dimethoxyethane (DME)(300 mL) and the mixture was ice-cooled. A solution of phosphonate{dimethyl (3R)-2-oxo-3-(m-tolyl)butylphosphonate} (54.7 g) synthesizedin Example 2 in DME (50 mL) was added, and the mixture was stirred for 1hr. To the above-mentioned solution was added a solution of(1S,5R,6R,7R)-6-formyl-7-benzoyloxy-2-oxabicyclo[3.3.0]octan-3-one (50.0g) in DME (400 mL), and the mixture was stirred for 1 hr. The reactionwas quenched with 350 mL of 10% brine, and the mixture was extractedwith ethyl acetate. The extract was dried over magnesium sulfate, andthe residue was concentrated under reduced pressure. The concentratedcrude product was recrystallized from t-butyl methyl ether to give thetitle compound (64.7 g). The structural property was as described below.

¹H-NMR (CDCl₃): δ 1.39 (d, J=7.0 Hz, 3H), 2.20-2.28 (m, 1H), 2.30 (s,3H), 2.34-2.41 (m, 1H), 2.49-2.57 (m, 1H), 2.76-2.85 (m, 3H), 3.80 (q,J=7.0 Hz, 1H), 5.03 (t, J=5.3 Hz, 1H), 5.23 (q, J=5.3 Hz, 1H), 6.19 (d,J=15.5 Hz, 1H), 6.69 (dd, J=15.6, 7.6 Hz, 1H), 6.94-7.19 (m, 4H),7.42-7.95 (m, 5H).

Example 4 Synthesis of(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-benzoyloxy-2-oxabicyclo[3.3.0]octan-3-one

A solution ofenone{(1S,5R,6R,7R)-6-[(1E,4R)-3-oxo-4-(m-tolyl)-1-pentenyl]-7-benzoyloxy-2-oxabicyclo[3.3.0]octan-3-one}(147.0 g) synthesized in Example 3 in THF (1480 mL) was cooled to −40°C., (−)-B-chlorodiisopinocampheylborane (1.7 M hexane solution) (721 mL)was added, and the mixture was stirred under ice-cooling for 20 hr.Acetone (183 mL) was added and the mixture was stirred for 3 hr. Aqueoussodium hydrogen carbonate was added, and the mixture was extracted witht-butyl methyl ether. The extract was dried over magnesium sulfate, andconcentrated under reduced pressure to give a crude title compound(649.9 g).

Example 5 Synthesis of(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxabicyclo[3.3.0]octan-3-one

The crude alcohol,{(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-benzoyloxy-2-oxabicyclo[3.3.0]octan-3-one}(649.9 g) synthesized in Example 4 was dissolved in methanol (740 mL),potassium carbonate (116.3 g) was added, and the mixture was stirred atroom temperature for 17 hr. Acetic acid was added to adjust to pH 7,methanol was evaporated, water was added, and the mixture was extractedwith ethyl acetate. The extract was purified by silica gel columnchromatography (hexane/ethyl acetate=4/1-0/1) to give the title compound(22.3 g). The structural property was as described below.

¹H-NMR (CDCl₃): δ 1.33 (d, J=7.0 Hz, 3H), 1.70 (s, 1H(OH)), 1.86 (ddd,J=11.3, 7.8, 3.2 Hz, 1H), 2.07 (d, J=4.4 Hz, 1H(OH)), 2.13-2.23 (m, 2H),2.34 (s, 3H), 2.35-2.44 (m, 3H), 2.47 (d, J=3.8 Hz, 1H), 2.56 (dd,J=18.2, 9.7 Hz, 1H), 2.80 (q, J=7.0 Hz, 1H), 3.79-3.85 (m, 1H),4.12-4.16 (m, 1H), 4.81 (dt, J=7.0, 3.2 Hz, 1H), 5.27 (ddd, J=15.7, 8.5,0.6 Hz, 1H), 5.50 (dd, J=15.2, 6.8 Hz, 1H), 6.94-7.20 (m, 4H).

Example 6 Synthesis of(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxabicyclo[3.3.0]octan-3-one

To a solution of the diol,{(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxabicyclo[3.3.0]octan-3-one}(988 mg) synthesized in Example 5 in N,N-dimethylformamide (DMF) (10 mL)were added at room temperature t-butyldimethylsilyl chloride (1.17 g)and imidazole (1.08 g), and the mixture was stirred for 2.5 hr. Thereaction mixture was poured into saturated aqueous sodium hydrogencarbonate, and the mixture was extracted with hexane/ethyl acetate=2/1mixture. The extract was dried over magnesium sulfate, concentratedunder reduced pressure and purified by silica gel column chromatography(hexane/ethyl acetate 20:1-10:1) to give the title compound (1.56 g).The structural property was as described below.

¹H-NMR (CDCl₃): δ−0.09 (d, J=6.4 Hz, 6H), 0.02 (d, J=2.4 Hz, 6H), 0.86(s, 9H), 0.89 (s, 9H), 1.27 (d, J=7.0 Hz, 3H), 1.86-1.92 (m, 1H),1.96-2.02 (m, 1H), 2.32 (s, 3H), 2.31-2.47 (m, 3H), 2.62-2.73 (m, 2H),3.82 (q, J=4.7 Hz, 1H), 4.05 (t, J=6.4 Hz, 1H), 4.86 (dt, J=8.0, 2.4 Hz,1H), 5.16 (dd, J=15.5, 7.4 Hz, 1H), 5.30 (dd, J=15.7, 6.3 Hz, 1H),6.90-7.16 (m, 4H).

Example 7 Synthesis of(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-one

Tetrahydrofuran (THF) (19 mL) was added to manganese bromide (1.48 g)and N-fluorobenzenesulfonimide (2.48 g), and the mixture was stirred for30 min, and cooled to −78° C. A solution of the lactone,{(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxabicyclo[3.3.0]octan-3-one}(0.5 g) synthesized in Example 6 in THF (5 mL) was added, a solution(0.5 M, 13 mL) of potassium bis(trimethylsilyl)amide in toluene wasadded and the mixture was warmed to 0° C. over 3.5 hr. The reactionmixture was poured into saturated aqueous sodium hydrogen carbonate, andthe mixture was extracted with hexane/ethyl acetate=1/1 mixture. Theextract was dried over magnesium sulfate, concentrated under reducedpressure and purified by silica gel column chromatography (hexane/ethylacetate 20:1) to give the title compound (0.32 g). The structuralproperty was as described below.

¹H-NMR (CDCl₃): δ−0.08-0.03 (m, 12H), 0.82 (s, 9H), 0.89 (s, 9H), 1.28(d, J=7.0 Hz, 3H), 1.70-1.77 (m, 1H), 1.96-2.04 (m, 1H), 2.31 (s, 3H),2.60-2.91 (m, 3H), 3.82-3.87 (m, 1H), 3.99-4.23 (m, 1H), 5.00 (t, J=6.4Hz, 1H), 5.06 (dd, J=15.7, 7.8 Hz, 1H), 5.33 (ddd, J=15.9, 6.7, 1.2 Hz,1H), 6.88-7.16 (m, 4H).

¹⁹F-NMR (CDCl₃): −113.1 (d, J=279.3 Hz), −91.0 (dd, J=279.3, 25.9 Hz).

Example 8 Synthesis of4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane

To a suspension of 4-(tetrazol-5-yl)butyltriphenylphosphonium bromide(14.0 g) in toluene (390 mL) was added a solution (0.5M, 120 mL) ofpotassium bis(trimethylsilyl)amide in toluene, and the mixture wasstirred at 60° C. for 1 hr. A solution of the difluorolactone,{(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-one}synthesized in Example 7 (4.32 g) in toluene (130 mL) was added at −10°C., and the mixture was stirred for 18 hr while warming the mixture toroom temperature. Aqueous sodium hydrogen carbonate was added to quenchthe reaction, and the mixture was extracted with hexane/ethylacetate=1/1 mixture. The extract was dried over magnesium sulfate,concentrated under reduced pressure and purified by silica gel columnchromatography (hexane/ethyl acetate=5/1-0/1) to give the title compound(4.1 g). The structural property was as described below.

¹H-NMR (CDCl₃): δ−0.14-0.01 (m, 12H), 0.82 (s, 9H), 0.89 (s, 9H),1.23-1.27 (m, 3H), 1.82-2.09 (m, 5H), 2.21-2.28 (m, 1H), 2.31 (s, 3H),2.45-2.53 (m, 1H), 2.64-2.73 (m, 2H), 2.93-2.97 (m, 2H), 3.90 (dd,J=11.7, 5.3 Hz, 1H), 4.08-4.09 (m, 1H), 4.84-4.87 (m, 2H), 5.27 (dd,J=15.5, 7.8 Hz, 1H), 5.44 (dd, J=15.6, 6.2 Hz, 1H), 6.92-7.16 (m, 4H).

¹⁹F-NMR (CDCl₃): −112.3 (d, J=253.4 Hz), −81.4 (dd, J=253.4, 18.7 Hz).

Example 9 Synthesis of4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane

THF (81 mL), water (81 mL) and acetic acid (244 mL) were added to thecompound (4.1 g) synthesized in Example 8, and the mixture was stirredat 35° C. for 46 hr. Water (500 mL) was added and the mixture wasextracted with chloroform. The extract was dried over magnesium sulfate,concentrated under reduced pressure and purified by silica gel columnchromatography (hexane/ethyl acetate=1/5-0/1) and recrystallized fromdiethyl ether to give the title compound (1.1 g). The structuralproperty was as described below.

¹H-NMR (CD₃OD): δ 1.30 (d, J=7.0 Hz, 3H), 1.69 (dddd, J=14.6, 7.6, 3.0,2.6 Hz, 1H), 1.82-1.95 (m, 2H), 2.10-2.16 (m, 2H), 2.29 (s, 3H),2.31-2.41 (m, 2H), 2.48-2.56 (m, 1H), 2.72 (q, J=7.0 Hz, 1H), 2.93 (t,J=7.6 Hz, 2H), 3.78 (q, J=7.6 Hz, 1H), 4.04-4.10 (m, 1H), 4.69 (dt,J=6.48, 2.96 Hz, 1H), 4.79 (dt, J=7.6, 5.0 Hz, 1H), 5.36-5.46 (m, 2H),6.95-7.13 (m, 4H).

¹⁹F-NMR (CD₃OD): −116.6 (d, J=250.5 Hz), −84.8 (ddd, J=251.9, 17.3, 14.4Hz).

Example 10 Synthesis of dimethyl 2-oxo-3-(m-tolyl)butylphosphonate

Using racemate of 2-(m-tolyl)propionic acid and in the same manner as inthe method of Examples 1-2, the title compound was synthesized. Thestructural property was as described below.

¹H-NMR (CDCl₃): δ 1.39 (d, J=7.2 Hz, 3H), 2.34 (s, 3H), 2.83 (dd,J=22.4, 14.4 Hz, 1H), 3.18 (dd, J=22.4, 14.0 Hz, 1H), 3.76 (dd, J=19.6,11.2 Hz, 6H), 3.99 (dd, J=14.0, 6.8 Hz, 1H), 7.01-7.27 (m, 4H).

Example 11 Synthesis of(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxabicyclo[3.3.0]octan-3-one

Using racemate of dimethyl 2-oxo-3-(m-tolyl)butylphosphonate and in thesame manner as in the method of Examples 3-6, the title compound wassynthesized. The structural property was as described below.

¹H-NMR (CDCl₃): δ −0.20-0.10 (m, 12H), 0.80-0.90 (m, 18H), 1.18-1.28 (m,3H), 1.85-2.20 (m, 2H), 2.31 (s, 3H), 2.30-2.80 (m, 5H), 3.80-4.15 (m,2H), 4.81-4.95 (m, 1H), 5.12-5.42 (m, 2H), 6.88-7.20 (m, 4H).

Example 12 Synthesis of(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-one

Using(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxabicyclo[3.3.0]octan-3-onesynthesized in Example 11 and in the same manner as in the method ofExample 7, the title compound was synthesized. The structural propertywas as described below.

¹H-NMR (CDCl₃): δ −0.20-0.05 (m, 12H), 0.80-0.90 (m, 18H), 1.19-1.29 (m,3H), 1.70-2.10 (m, 2H), 2.31 (s, 3H), 2.60-3.05 (m, 3H), 3.84-4.12 (m,2H), 4.95-5.50 (m, 3H), 6.85-7.20 (m, 4H).

¹⁹F-NMR (CDCl₃): −113.6-−112.8 (m), −91.7-−90.6 (m).

Example 13 Synthesis of4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane

Using(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-onesynthesized in Example 12 and in the same manner as in the method ofExample 8, the title compound was synthesized. The structural propertywas as described below.

¹H-NMR (CDCl₃): δ −0.15-0.05 (m, 12H), 0.80-0.89 (m, 18H), 1.20-1.28 (m,3H), 1.80-3.05 (m, 14H), 3.90-4.15 (m, 2H), 4.85-4.95 (m, 2H), 5.23-5.58(m, 2H), 6.90-7.20 (m, 4H).

¹⁹F-NMR (CDCl₃): −113.0-−111.3 (m), −82.0-−80.7 (m).

Example 14 Synthesis of4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane

Using4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butanesynthesized in Example 13 and in the same manner as in the method ofExample 9, the title compound was synthesized. The structural propertywas as described below.

¹H-NMR (CDCl₃): δ 1.15-1.35 (m, 3H), 1.80-3.00 (m, 11H), 2.29 (s, 3H),4.05-4.20 (m, 2H), 4.75-4.85 (m, 2H), 5.35-5.70 (m, 2H), 6.95-7.25 (m,4H).

¹⁹F-NMR (CDCl₃): −114.5-−112.7 (m), −83.5-−81.8 (m).

Example 15 Synthesis of5-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]pentanoicacid (carboxylate form)

Using(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-t-butyldimethylsiloxy-4-(m-tolyl)-1-pentenyl]-7-t-butyldimethylsiloxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-onesynthesized in Example 12 and (4-carboxybutyl)triphenylphosphoniumbromide, and in the same manner as in the method of Examples 8-9, thetitle compound was synthesized. The structural property was as describedbelow.

¹H-NMR (CD₃OD): δ 1.17-1.30 (m, 3H), 1.63-2.79 (m, 11H), 2.29 (s, 3H),3.75-4.12 (m, 2H), 4.66-4.85 (m, 2H), 5.40-5.58 (m, 2H), 6.95-7.15 (m,4H).

¹⁹F-NMR (CD₃OD): −118.3-−117.7 (d, J=250.4 Hz), −86.1-−85.3 (m).

Example 16 In Vitro Metabolic Stability of the Compound of the PresentInvention

A mixture of the compound F and compound J of the present inventiondescribed in Table 1 (F:J=52:41, synthesized in Example 14), and amixture of compounds wherein the tetrazole groups at C-1 of compound Fand compound J are respectively substituted by carboxylic acid (referredto as carboxylate form, F:J=54:34, synthesized in Example 15) weretested.

First, a mitochondria fraction was prepared from the rat liver accordingto the following Reference A. Then, in reference to the method ofYAMAGUCHI et al. described in the following References B and C, anNADPH-independent β oxidation reaction was studied. The reaction wascarried out at 37° C. for 30 min, and stopped with a methanol solutioncontaining a suitable internal standard substance. Each compound wasquantified by the internal standard method using a high performanceliquid chromatography mass spectrometry apparatus (LC-MS/MS). Thecompound residual ratio after metabolic reaction of compounds F, J andeach carboxylate form thereof in rat mitochondria fraction is shown inthe following Table 2 in average±standard deviation of 3 experiments.

TABLE 2 Residual Ratio of Parent Compound after β-Oxidation ReactionCompound Residual ratio (%) Compound F 91.6 ± 6.8 Compound J 90.1 ± 6.9Carboxylate form of Compound F 27.8 ± 2.2 Carboxylate form of Compound J44.1 ± 2.1

As is clear from the above-mentioned Table 2, representative compound Fand compound J of the present invention are not subject to β oxidationin a mitochondria fraction.

REFERENCES

-   A) The Japanese Biochemical Society, ed., Biochemical Experiment    Course 12 energy metabolism and biological oxidation (vol. 1), Tokyo    Kagaku Dojin, p. 217-218, 1st ed. 2nd printing, published on Jul.    11, 1979.-   B) Drug Metabolism And Disposition 23(11): 1195-1201 (1995).-   C) Xenobiotica 26(6): 613-626 (1996).

Example 17 Plasma Pharmacokinetics after Intravenous Administration toRats

To verify the in vivo metabolic stability of the compound of the presentinvention, plasma pharmacokinetics was evaluated after intravenousadministration to rats. Male rats (6 weeks old, body weight 160-180 g)were acclimated for 1 week, and the animals diagnosed healthy were used.A mixture of compound F and compound J of the present inventiondescribed in Table 1 (F:J=52:41), and a mixture of carboxylate forms ofcompound F and compound J (F:J=54:34), and compound F (synthesized inExample 9) were dissolved in a small amount of ethanol and physiologicalsaline was added to prepare test compound solutions. The test compoundsolutions were instantaneously administered intravenously at 1 mL/kgfrom the femoral vein of non-fasting rats under light ether anesthesia.Venous blood was drawn from the tail vein 5, 15, 30, 45, 60, 90 and 120min after administration. The blood was mixed with heparin andcentrifuged (3000 rpm, 4° C., 15 min) to obtain plasma. The plasmacompound concentration was determined by the internal standard methodusing LC-MS/MS. The determination range by this method was from 0.1 to100 ng/mL. The compound concentrations obtained from each rat wereanalyzed in a model-independent way using a pharmacokinetics analysissoftware WinNonlin (ver. 3.3), and average±standard deviation of 3animals for each group was obtained. The apparent half-life (t_(1/2)) inthe elimination phase is shown in the following Table 3.

TABLE 3 Apparent Half-life of Elimination Phase of Compounds afterIntravenous Administration to Rats test compound dose t_(1/2) (min)compound F administered as 50 μg/kg (mixture 115 ± 31  a mixture withisomers with isomers) compound J administered as 50 μg/kg (mixture 77 ±19 a mixture with isomers with isomers) compound F 300 μg/kg 158 ± 15 carboxylate form of 50 μg/kg (mixture 9.6 ± 1.7 compound F administeredas with isomers) a mixture with isomers carboxylate form of 50 μg/kg(mixture 8.9 ± 0.3 compound J administered as with isomers) a mixturewith isomers

As is clear from the above-mentioned Table 3, t_(1/2) values of thecompound F and compound J of the present invention were about 1-2 hr,which were markedly prolonged in comparison with less than 10 min of thecarboxylate forms. It suggests that the compound F and compound J of thepresent invention have excellent metabolic stability.

Example 18 Receptor Affinity

PG receptor affinity of compound F and compound J of the presentinvention was evaluated. Compound F used was one synthesized in Example9, and compound J was prepared by separating and purifying the compoundsynthesized in Example 14 with a column (the same preparations were usedin the following Examples). COS-7 cells were transfected with the genesof mouse EP4, human EP1-4 or human IP to overexpress the receptor andthen the cell membranes were collected. As a labeled ligand,tritium-labeled PGE₂ was used for EPs; tritium-labeled iloprost, for IP.The dissociation constant Kd value was obtained and inhibition constantKi value of each test compound was determined in a conventional method.As a reference control, PGE₂ was used for EPs; beraprost sodium (BPS),for IP.

As a result, the dissociation constant was almost identical to theliterature values. As shown in Table 4, PGE₂ equivalently bound toEP1-4, and its selectivity for EP subtypes was not observed. BPSselectively bound to IP. Compounds F and J bound to mouse and humanEP4s. The affinity of compound F for binding to human EP4 was higher by60-fold or more than those to human EP1, EP2 and EP3 receptors, and100-fold or more than that to IP. The binding affinity of compound J tohuman EP4 was 40-fold higher than that to human IP.

TABLE 4 Binding Affinity of Test Compounds to Various Receptors Ki value(nmol/L) Test Mouse Human Human Human Human Human Compound EP4 EP1 EP2EP3 EP4 IP PGE₂ 2.6 2.9 6.0 2.1 1.1 — BPS — 942 4989 946 6148 160Compound F 68 610 570 410 6.6 670 Compound J 590 4600 5400 1500 83 3400

Example 19 Agonist Activity

The EP4 agonist activity of compound F and compound J of the presentinvention was evaluated in a conventional method. In brief, human EP4gene and CRE-LUC reporter gene were transfected into COS-7 cells. Thecells were treated with a test compound one day later, and incubated for3 hr. The cells were washed, a luminescence substrate was added, and theluminescence intensity was measured for the agonist activity. As areference control, PGE₂ was used and the maximum activity of PGE₂ wastaken as 100%. The 50% effective concentration (EC₅₀) was calculated andthe agonist activity of the test compound was compared.

As a result, as shown in Table 5, compounds F and J are EP4 agonists.

TABLE 5 EP4 Agonist Activity of Test Compounds EC₅₀ (nmol/L) TestCompound EP4 PGE₂ 0.68 BPS >1000 Compound F 3.33 Compound J 440Each value is a geometric mean of three determinations.

Example 20 Inhibitory Effect on Platelet Aggregation

The inhibitory effect of compounds F and J, and carboxylate formsthereof on platelet aggregation was evaluated. The carboxylate formswere prepared by separating and purifying the compound synthesized inExample 15 with a column. Blood was drawn from 3 healthy volunteersusing sodium citrate as an anticoagulant, and platelet-rich plasma wasprepared. The platelet-rich plasma was treated with physiological salineor test compounds and, 2 min later, platelet aggregation was inducedwith adenosine diphosphate (ADP, final concentration 10 μmol/L) andrecorded with a platelet aggregation measurement apparatus(nephelometry). The maximum aggregation of the group treated withphysiological saline was taken as 100%, the concentration required for50% inhibition (IC₅₀) thereof was calculated, and the inhibitory effectwas assessed.

As a result, as shown in Table 6, compounds F and J had a considerablyweak inhibitory effect on aggregation as compared to the IP agonists(BPS and carboxylate forms).

TABLE 6 Inhibitory Effect of Test Compounds on Human PlateletAggregation Test Compound IC₅₀ (nmol/L) BPS 36 Compound F 917 Compound J2725 Carboxylate form of Compound F 120 Carboxylate form of Compound J190Each IC₅₀ value is a geometric mean of three volunteers.

Example 21 Effect on Blood Pressure and Heart Rate in Mice

Male ICR mice (5 weeks old, Japan SLC) were purchased, acclimated for 6days and used for the test. The animals were treated and measured underisoflurane inhalation anesthesia (anesthesia introduced at 2.5%,maintained at 1.8-2.1%), while body temperature was kept at 37° C. witha Heat Controller (ATC-402, Unique Medical Co., Ltd.). A catheter foradministration of test compounds was inserted into the left femoralvein, and another catheter was inserted into the right femoral arteryand connected to a pressure transducer to record each parameter ofhemodynamics.

Compound F, the carboxylate form of compound F and BPS wereintravenously administered at a dose of 0.01 mg/5 mL/kg, and the effecton the mean blood pressure and heart rate was evaluated with computersoftware for the analysis of hemodynamics (Fluclet, Dainippon SumitomoPharma Co., Ltd.). To the control group was intravenously administered asolvent (1.2% ethanol solution) at 5 mL/kg in the same manner. Theresults are expressed as a ratio of change in each parameter before andafter the drug administration. Three to 6 animals were used for eachgroup, and the results were expressed as average±standard deviation.

As a result, the solvent did not affect the mean blood pressure, but thecarboxylate form of compound F and BPS remarkably decreased the bloodpressure 1 min after the administration, at which time the maximumdecrease ratio was 45% and 38%, respectively (FIG. 1A). The decreasedblood pressure recovered in 10 min, but the heart rate was on theincrease and remained high even after 10 min (FIG. 1B). The maximumdecrease ratio by compound F was 16.2%, which was not significant (FIG.1A). In addition, compound F had little effect on the heart rate (FIG.1B). Therefore, the effect of compound F on the blood pressure and heartrate was extremely weak, as compared to the carboxylate form and BPS (IPagonist).

Example 22 Suppressive Effect on Inflammatory Cytokine Production

Using human peripheral blood, an anti-inflammatory effect of compounds Fand J in vitro was studied. Blood was collected from 3 healthyvolunteers, and CD4 positive T cells were prepared. Anti-CD3 antibodyand anti-CD28 antibody were added, and 24 hr later the amounts of IL-2and TNFα released in the medium were measured by ELISA. In addition, thecollected whole blood was diluted with the medium, treated withindometacin to inhibit production of endogenous PGE2, and added withlipopolysaccharide. The amount of IP-10 released into the medium for 48hr was measured with ELISA. In both cases, the test compound was added30 min before stimulation. The production amount of the solvent controlgroup was taken as 100% and the concentration required for 50%inhibition (IC₅₀) thereof was determined.

As a result, as shown in Table 7, PGE₂ and compound F stronglysuppressed the production of inflammatory cytokines IL-2, TNFα and IP-10even at an extremely low concentration. Although weak as compared tocompound F, compound J also suppressed the cytokine production. Theeffect of each compound reflects its EP4 affinity and EP4 agonistactivity.

As mentioned above, even though the compounds of the present inventionincluding compound F are PGI₂ derivatives, they are EP4 selectiveagonists with an extremely reduced IP agonist activity compared with theactivity observed in the carboxylate forms at C-1. The compound of thepresent invention is expected to show similar clinical efficacy in thedisease group for which an EP4 agonist is effective. In contrast, thecompound causes less concern of the side effects such as bleeding,hypotension, cardiac palpitation and face flush, since the effectthereof on the circulatory system due to the IP agonist action is alsoweakened. For example, it is important to attenuate such IP agonistactions in treatment of inflammatory bowel disease with intestinalbleeding. Using compound F, the efficacy of the compound of the presentinvention was determined in various animal models such as inflammatorybowel disease.

TABLE 7 Suppressive Effect of Test Compounds on Cytokine Production TestIC₅₀ (nmol/L) Compound IL-2 TNF-α IP-10 PGE₂ 0.062 0.447 0.168 CompoundF 0.509 1.254 1.144 Compound J 64.0 89.5 102Each value is a geometric mean of three volunteers.

Example 23 Prophylactic Effect on Dextran Sodium Sulfate-Induced ColitisModel in Mice

The prophylactic effect of compound F on ulcerative colitis was examinedin dextran sodium sulfate-induced colitis model. The animal modeldisplays inflammation localized to the large intestine, resulting indiarrhea and blood feces, which resembles pathologic condition of theclinical ulcerative colitis closely (cf.: References D and E).

Female BALB/c mice (6 weeks old, Japan SLC) were purchased, acclimatedfor 1 week and used for the study. Except the normal group, the micewere allowed to freely drink a dextran sodium sulfate (to be abbreviatedas DSS, MP Biochemicals, M.W. 36,000-50,000, Lot No. 3439J) solutionprepared to 2.2 w/v % for 9 days to induce colitis. Compound F wasorally administered at doses of 0.1, 0.3 and 1 mg/kg, once a day, daily,from the start day of DSS drinking (day 0) to one day before autopsy(day 9). To the control group was orally administered a solvent (1 vol %ethanol solution) at 10 mL/kg in the same manner.

Our preliminary study had revealed that mouse feces show a correlationbetween the water content and shape thereof. Thus, to determine thelevel of diarrhea, the stool was graded into 6 levels; normal (score 0),spherical stool being not less than 50% (score 1), banana-shaped stoolbeing less than 50% (score 2), banana-shaped stool being not less than50% (score 3), muddy stool (score 4), watery stool (score 6) (stoolconsistency score). The fecal occult blood (including proctorrhagia) wasgraded using fecal occult blood slide 5 Shionogi II (Shionogi & Co.,Ltd.) into 6 levels; negative (no change of the slide color from yellow,score 0), weakly positive (slightly blue green, score 1), positive (bluegreen, score 2), moderately positive (clear blue green, score 3),strongly positive (instantaneous color change to dark blue with colordeveloper, score 4), and proctorrhagia (score 5). The sum of the stoolconsistency score and occult blood score was defined as the stool score.Eight to 10 animals were used for each group, and the results wereexpressed as average±standard deviation. On the day of autopsy, afterlaparotomy under ether anesthesia and blood collection, the mice wereexsanguinated to death. Then the large intestines were dissected fromjust below the cecum to the anus and the length thereof was measured.

As a result, the body weight gradually increased over the study periodwithout any difference among groups. The control group showed obviousloose stools and occult blood in stools from day 4 of DSS drinking. Onthe day of autopsy (day 9), the length of the large intestine thereofwas clearly shorter than that of the normal group. Compound Fdose-dependently suppressed the increase in the stool score, which was asuppressive tendency at 0.1 mg/kg and significant at 0.3 and 1 mg/kg(FIG. 2A). Likewise, compound F showed a dose-dependent suppressiveeffect on shortening of the large intestine (FIG. 2B). Thus, compound Fclearly prevented the onset of ulcerative colitis.

REFERENCES

-   D) Lab. Invest. 69(2): 238-249 (1993).-   E) Inflamm. Res. 45(4): 181-191 (1996).

Example 24 Effect of IP Agonist on Dextran Sodium Sulfate-InducedColitis Model in Mice

Whether or not an IP agonist has an effect on such colitis model wasdetermined using a selective IP agonist, BPS.

Female C57BL/6 mice (6 weeks old, Japan SLC) were purchased, acclimatedfor 1 week and used for the study. Except the normal group, the micewere allowed to freely drink a 3 or 2 w/v % DSS (MP Biochemicals, LotNo. 5653H and 5464H, respectively) solution for 1 week to inducecolitis. BPS at a dose of 0.3 mg/kg and compound F at doses of 0.3 and 1mg/kg were orally administered once a day, daily, from the start day ofDSS drinking (day 0) to one day before autopsy (day 9). To the controlgroup was orally administered a solvent (1 vol % ethanol solution) at 10mL/kg in the same manner. The consistency of the stool was graded fromscore 0 to 4, with normal (0), partly loose stool (1), loose stool (2)and diarrhea (4). The blood feces was also graded from score 0 to 4,with normal (0), partly blood feces (1), blood feces (2) and blood fecesplus proctorrhagia (4). The sum of both grades was defined as the stoolscore (maximum 8). Furthermore, the length of the large intestine wasmeasured in the same manner as in Example 23. Six to 10 animals wereused for each group, and the results are shown in average±standarddeviation.

As a result, the IP agonist BPS was not effective, but rather showed atendency toward aggravation of the stool score. Also, it showed noeffect on the shortening of the large intestine (FIGS. 3A and 3C).However, compound F demonstrated a superior prophylactic effect on theonset of colitis in the same manner as seen in Example 23 (FIGS. 3B and3D). Thus, the treatment effect is brought by an EP4 agonist action, andis sometimes weakened by an IP agonist action. As such, being aselective EP4 agonist is important.

Example 25 Prophylactic Effect on Dextran Sodium Sulfate-Induced Colitisin Rats

Prophylactic effect of compound F on colitis was also studied in rats.Male SD rats, 7 weeks old, body weight around 210 g-240 g (CharlesRiver) were purchased, acclimated for 1 week and used for the study.Except the normal group, the rats were allowed to freely drink a DSS (MPBiochemicals, M.W. 36,000-50,000, Lot No. 4556J) solution prepared to5.5 w/v % for 8 days to induce colitis. Compound F at doses of 0.3, 1and 3 mg/kg was orally administered once a day, daily, from one daybefore the start day of DSS drinking to one day before autopsy (day 7).To the control group was orally administered a solvent (1 vol % ethanolsolution) at 5 mL/kg.

On day 8 from the start of DSS drinking, 1.25 w/v % Evans blue solutionwas administered at 0.2 mL/100 g from the tail vein. After 30 min, therats were subjected to laparotomy under ether anesthesia andexsanguinated to death. Thereafter, the large intestine was dissectedfrom just below the cecum to the anus, and the length was measured witha scale. After the contents of the large intestine were removed, thecolonic tissue of 7 cm long from the anus was washed 3 times withphysiological saline and dried overnight with a vacuum pump. The nextday, the dry weight was measured, formamide (2 mL) was added, the dyewas extracted at 50° C. overnight, and the level thereof was measured at620 nm. A standard curve was prepared using an Evans blue standardsolution, and the amount (mg) of Evans blue in 1 g of the colonic tissuewas calculated to estimate degree of colonic tissue injury.

To show the level of diarrhea, the shape of stool was graded into 6levels, with normal (score 0), rod-like stool being less than 50% (score1), rod-like stool being not less than 50% (score 2), rod-like stool andpartly muddy stool (score 3), muddy stool (score 4) and watery stool(score 6) (stool consistency score). Fecal occult blood was graded bythe same method described in Example 23 (occult blood score). The sum ofstool consistency score and occult blood score was defined as the stoolscore. Seven to 10 animals were used for each group, and the results areshown in average±standard deviation.

As a result, the body weight of the control group gradually increasedconsistently, but the increase was significantly smaller than that ofthe normal group. The stool score of the control significantly elevatedfrom day 1 of DSS drinking. On the day of autopsy (day 8), the largeintestine thereof showed an apparent tissue injury and a significantshortening. In contrast, administration of compound F at 1 mg/kg and 3mg/kg showed a suppressive tendency or significant suppressive effect onthese events (FIGS. 4A, 4B, 4C). That is, compound F prevents ulcerdevelopment in the large intestine and normalizes the organ function,thereby leading suppression of symptoms of diarrhea and blood feces.

Example 26 Therapeutic Effect on Remission/Relapse Model of DextranSodium Sulfate-Induced Colitis in Mice

Next, therapeutic effect of compound F on colitis was studied in achronic model. Female BALB/c mice, 6 weeks old, body weight about 20 g(Japan SLC) were purchased, acclimated for 1 week and used for thestudy. The mice were divided into a colitis induction group and a normalgroup. The colitis induction group was allowed to freely drink a 2.6 w/v% DSS (MP Biochemicals, M.W. 36,000-50,000, Lot No. 4556J) solution toinduce colitis. On day 8 when the stool score (defined in Example 23) ofthe colitis induction group reached about 4.5, the mice were subdividedinto a control group, a compound F 1 mg/kg administration group and asalazosulfapyridine (SIGMA, Lot No. 085K1930, hereinafter to beabbreviated as SASP) 100 mg/kg administration group. Then the mice wereallowed to freely drink distilled water instead of DSS solution for 9days (remission period). After the grouping, the stool score wasevaluated every 3-4 days. When the score of the control group reachedabout 1, the mice were again allowed to drink the DSS solution to causea relapse (relapse period). The periods of remission and relapse weretaken as 1 cycle and the cycle was repeated 5 times. As for the 5thcycle, however, only the remission period was performed.

Compound F at a dose of 1 mg/kg and SASP at a dose of 100 mg/kg wereorally administered once a day, daily, for 50 days from the initialremission period (day 8 from the start of 2.6 w/v % DSS drinking) to thefifth remission period (day 57 from the start of 2.6 w/v % DSSdrinking). To the control group was orally administered a solvent (1 vol% ethanol solution) at 10 mL/kg. If a mouse had score 0 of both stoolconsistency score and occult blood score on the last day of eachremission period, the mouse was regarded as “in remission”. Theremission ratio (%) was calculated as a ratio of mice in remission ineach group. Eight to 10 mice were used for each group and the resultsare shown in average value.

As a result, the stool score of the control group increased in therelapse period, and decreased in the remission period. The score wassignificantly higher than that of the normal group almost throughout thestudy period (FIG. 5). The remission ratio thereof was 35.5% on averageof 5 remission periods (Table 8). Compound F decreased the stool scoreearly in the remission period, and suppressed an increase in the scorein the relapse period. The remission ratio thereof was not less than 60%in any remission period, and the average was 66.0%, which was evidentlyhigher than that of the control group. On the other hand, SASP did notshow a clear effect on the stool score in either the remission period orthe relapse period. The remission ratio thereof was slightly higher inthe 1st, 3rd and 4th cycles than that of the control group, converselylower in the 2nd and 5th cycles, and the average value was equivalent tothat of the control group.

As shown above, compound F provides not only a prophylactic effect butalso a therapeutic effect, as well as a remission maintaining effect.Moreover, the effects thereof are considered to be far superior to SASPin clinical use.

TABLE 8 Remission Ratio of Remission/Relapse Model of DSS-inducedColitis in Mice Remission Ratio (%) Number of Cycle Cycle Cycle CycleCycle Treatment animals 1 2 3 4 5 Average Control 9 33.3 66.7 11.1 33.333.3 35.5 Compound F 10 60.0 80.0 70.0 60.0 60.0 66.0 1 mg/kg SASP 850.0 50.0 37.5 50.0 12.5 40.0 100 mg/kg

Example 27 Prophylactic Effect on CD4⁺CD25⁻ T Cell Transfer ColitisModel in Mice

The effect on Crohn's disease, another type of inflammatory boweldisease, was studied. T cell transfer model is well known as a Crohn'sdisease model, which develops chronic gastritis or enteritis (see:References F, G, H). In addition, it can also be regarded as an animalmodel of intestinal Behcet's disease or simple ulcer, suffering fromsimilar intestinal ulcer accompanied by activation of T cells (see:References I, J).

Female BALB/cA Jcl mice, 6 weeks old, body weight 19-23 g (CLEA Japan,Inc.) and female C.B-17/Icr-scid mice (6 weeks old, CLEA Japan, Inc.)were purchased, acclimated for 1 week and used for the study.

After laparotomy under ether anesthesia, BALB/cA Jcl mice wereexsanguinated to death through the abdominal aorta and caudal vena cava,and the spleen was isolated. Splenocytes were prepared from the spleenand then CD4⁺CD25⁻ T cells were prepared with a CD4⁺ T cell IsolationKit (No. 130-090-860, Milky Biotech Co., Ltd.) and CD25-Biotin antibody(No. 130-092-569, Milky Biotech Co., Ltd.). The cells were separatedusing the autoMACS Separator (Milky Biotech Co., Ltd.). The separatedCD4⁺CD25⁻ T cells were suspended in physiological phosphate buffersolution, and 2.5×10⁵ cells per animal were intraperitoneallyadministered to C.B-17/Icr-scid mice to induce colitis.

One mg/kg of compound F or prednisolone was initially administered at 5hr before transfer of CD4⁺CD25⁻ T cells, and orally administeredthereafter once a day, daily, for 20 days. To the control group wasorally administered a solvent (1 vol % ethanol solution) at 10 mL/kg. Aclinical endpoint was the sum of stool consistency score (0-5), fecaloccult blood score (0-4) and body weight decrease score (0-5), termed asthe Disease Activity Index score (hereinafter to be abbreviated as DAIscore: highest score 14). The stool consistency score was graded for thehardness of stool as normal (0), slightly loose (1), somewhat loose (2),loose (3), considerably loose (4) and diarrhea (5). The fecal occultblood score was evaluated in the same manner as in Example 23. The bodyweight decrease score was graded for the changes in the body weight asincrease (0), decrease of less than 3% (1), decrease of not less than 3%and less than 6% (2), decrease of not less than 6% and less than 9% (3),decrease of not less than 9% and less than 12% (4), and decrease of notless than 12% (5). Eight to 10 mice were used for each group and theresults were expressed as average.

As a result, stool consistency score and fecal occult blood score of thecontrol group showed a clear increase from 12 days after T cell transferand the body weight decrease score showed a clear increase on day 19,all reaching almost maximum 21 days later. Compound F suppressed theincreases in both the stool consistency score and the fecal occult bloodscore by almost half as shown in FIGS. 6A and 6B, respectively, andprevented the increase in the body weight decrease score almostcompletely as shown in FIG. 6C. On the other hand, though prednisolonesuppressed an increase in the fecal occult blood score by almost thesame level as the compound F administration as shown in FIG. 6B, itfailed to show a clear effect on the stool consistency score on day 21as shown in FIG. 6A. In addition, the body weight decrease scoreremained at higher values than those in the control group over the studyperiod as shown in FIG. 6C, and prednisolone clearly worsened the score.As shown in FIG. 6D, the DAI score indicated that compound F iscomprehensively superior to prednisolone.

Therefore, compound F can suppress the condition of Crohn's disease,intestinal Behcet's disease and simple ulcer as well as ulcerativecolitis more effectively than existing drugs.

REFERENCES

-   F) Immunol Rev. 182: 190-200 (2001).-   G) Int. Immunopharmacol. 6(8): 1341-1354 (2006).-   H) J. Immunol. 160(3): 1212-1218 (1998).-   I) Clin. Exp. Immunol. 139(2): 371-378 (2005).-   J) Histopathology. 45(4): 377-383 (2004).

Example 28 Effect on Ethanol-Induced Gastric Mucosal Injury Model inRats

The suppressive effect of compound F on gastric mucosal injury wasinvestigated in ethanol-induced gastric mucosal injury model in rats.This model is frequently used as an animal model of human acutegastritis associated with congestive mucosal injury (Reference K).

Male SD rats (7 weeks old, Charles River) were purchased throughOriental BioService Inc., acclimated for 1 week and used for the study.The rats were grouped based on the body weight, placed in a clean cageset with a wire mesh floor one day before the study, fasted for 19 hr(without water for last 3 hr), and orally administered with ethanol(special grade, Nacalai Tesque, Lot No. V8A5862, 1.5 mL) in all groupsto induce gastric mucosal injury. Compound F was orally administered atdoses of 0.01, 0.1 and 1 mg/kg 30 min before induction of gastricmucosal injury at a volume of 5 mL/kg. To the control group was orallyadministered a solvent (1 vol % ethanol solution) at 5 mL/kg in the samemanner. Eight animals were used for each group.

The rats were bled to death from the abdominal aorta and caudal venacava under ether anesthesia after 1 hr from the ethanol administration,and the stomach was isolated. The isolated stomach was immediatelyfilled with 2 vol % neutral formalin solution (6 mL) and fixed for 15min. The stomach was incised along the midline of the greater curvaturefrom the cardiac part to the pyloric part, and extended on a vinylchloride board. The length and width of each ulcer were measured under astereomicroscope, the area was calculated, and the sum thereof was takenas the total ulcer area.

As a result, total ulcer area of the control group averaged 103 mm².Compound F significantly reduced the total ulcer area in adose-dependent manner from 0.01 mg/kg, and almost completely reduced thearea at a dose of 1 mg/kg (FIG. 7). Thus, compound F suppressed thegastric mucosal injury.

REFERENCE

-   K) Dig Dis Sci. 31(2 Suppl), 81S-85S (1986).

Example 29 Effect on Indomethacin-Induced Small Intestinal Injury Modelin Rats

The suppressive effect of compound F on small intestinal injury wasinvestigated using indomethacin-induced small intestinal injury model inrats. Administration of non-steroidal anti-inflammatory drugs (NSAIDs)is known to induce hemorrhagic injury in the small intestine of human.This model is characterized by mucosal injury of the small intestineinduced by administration of a NSAID, indomethacin, and shows pathologysimilar to that of NSAIDs-induced small intestinal injury in patients orCrohn's disease (References L and M).

Male SD rats, 7 weeks old (Charles River) were purchased, acclimated for1 week and used for the study. The rats were grouped based on the bodyweight and subcutaneously administered with indomethacin (SIGMA, Lot No.19F0018) at 15 mg/5 mL/kg to all groups to induce small intestinalinjury. Compound F at doses of 0.01, 0.1 and 1 mg/kg was orallyadministered at a volume of 5 mL/kg 30 min before and 6 hr after thesubcutaneous administration of indomethacin. To the control group wasorally administered a solvent (1 vol % ethanol solution) at 5 mL/kg inthe same manner. Eight animals were used for each group.

The rats were intravenously administered with 2 mL of 10 mg/mL Evansblue solution under ether anesthesia 23.5 hr after the indomethacinadministration. After 30 min, the rats were bled to death from theabdominal aorta and caudal vena cava under ether anesthesia and thesmall intestine was isolated. The isolated small intestine was filledwith an adequate amount (about 35 mL) of 2 vol % neutral formalinsolution, and fixed for about 15 min. Thereafter, the small intestinewas incised along the mesenteric attachment site, and extended on avinyl chloride board. The length and width of each ulcer were measuredunder a stereomicroscope, the area was calculated, and the sum thereofwas taken as the total ulcer area.

As a result, the total ulcer area in the small intestine was about 730mm² in the control group. In contrast, the compound F administrationgroup significantly reduced the ulcer area in a dose-dependent mannerfrom a dose of 0.1 mg/kg administration, and completely reduced the areaat a dose of 1 mg/kg (FIG. 8). Thus, compound F strongly suppressed thesmall intestinal mucosal injury.

REFERENCES

-   L) Aliment Pharmacol Ther. 7(1), 29-39 (1993).-   M) Acta Gastroenterol Belg. 57(5-6), 306-309 (1994).

From the above, compound F showed a superior suppressive action on thedirect injury to the gastrointestinal tract mucosa due to alcohol andthe like and mucosal regenerative failure due to NSAIDs and the like.Therefore, compound F is expected to show a protective effect and atissue repair effect on mucosal injury of the gastrointestinal tract.

As shown in the above-mentioned examples and found with compound F, thecompound of the present invention is effective for gastrointestinaltract injury and delay in cure due to immune-related inflammation ofdigestive tract, drug-induced mucosal injury of the gastrointestinaltract and drug-induced mucosal regenerative failure. Specifically, it isuseful for inflammatory bowel disease such as ulcerative colitis andCrohn's disease, alcoholic gastritis or gastric ulcer, small intestinalulcer and the like. These actions are based on an EP4 agonist action,and are not limited to the recited diseases.

Example 30 Effect on Anti-Thy-1 Antibody-Induced GlomerulonephritisModel in Rats

Male Slc: Wistar rats (6 weeks old, Japan SLC) were purchased,acclimated for 1 week and used for the study. Except the normal group,anti-Thy-1 antibody (mouse anti-CD90 antibody (UK-Serotech Ltd.Code:MCA47XZ, clone No: MRC OX-7, Lot No. 0303)) was administered onceintravenously to the animals. A mixture of compound F and compound J(F:J=52:41, indicated as compound F/J) was orally administered from theday of the antibody administration (day 0) to day 6, daily, twice a day(morning and evening; each 0.3 mg/kg). After 3 days from the antibodyadministration, urine was collected for one day. On day 7 of theantibody administration, the animal was autopsied. The right kidney wasisolated, weighed and fixed with formalin. Five to 8 animals were usedfor each group and the results were expressed as average±standarddeviation.

As a result, body weight gain was suppressed in the control group ascompared to the normal group after 1 day from the antibodyadministration. However, the body weight increased like the normal groupafter 3 days. The body weight of compound F/J-treated group showed ashift similar to that of the control group. The 24-hr urine volume ofthe control group increased as compared to the normal group. However,that of the compound F/J-treated group was of the same level as thenormal group (FIG. 9A). The 24-hr urine protein markedly increased inthe control group, whereas a lower value than control was found in thecompound F/J-treated group (FIG. 9B). The relative kidney weightmarkedly increased in the control group, but a value lower than controland near the normal was observed in the compound F/J-treated group (FIG.9C). Renal histopathological evaluation revealed that the control groupremarkably increased the total glomerular cells, mesangial region andPCNA-positive cells in the glomerulus. The compound F/J-treated groupsignificantly suppressed the increase in all of these measures (FIGS.9D, 9E, 9F).

Therefore, the compound of the present invention normalizes the urinevolume, decreases proteinuria and suppresses the immune reaction andgrowth reaction of glomerulus, indicating that it is effective fornephritis.

Example 31 Effect on Intraocular Pressure in Rabbits

Japanese white rabbits (male, 10 weeks old, BIOTEC Co., Ltd.) werepurchased, acclimated for 1 week and used for the study. Compound Fsolution (0.01 w/v %) was instilled once into the cornea of both eyes ata volume of 50 μL/eye with a micropipette. The intraocular pressure ofthe right eye was measured and the local irritative effect on the eyewas evaluated using the left eye. After surface anesthesia withoxybupranol (Benoxil 0.4% instillation solution), the intraocularpressure was measured before ocular instillation and 1, 2, 3, 4, 6 and 8hr after the instillation using a pneumatonometer (Alcon Ltd.). Inaddition, the local irritative effect on the eye was evaluated byscoring conjunctival congestion, conjunctival edema, cornea opacity,iris congestion, excretion and eye closure performance. For theevaluation, phosphate buffer was used for the solvent control group, andisopropyl unoprostone (Rescula eye drops, Santen Pharmaceutical Co.,Ltd.) was used for comparison. Six animals were used for each group andthe results were expressed as average.

As a result, the intraocular pressure of the solvent control groupshifted within the range of ±2 mmHg from the time 0 to 8 hrs after theinstillation. The compound F group showed a decrease of 6.9 mmHg inintraocular pressure 1 hr after the instillation, of 9.3 mmHg 2 hrafter, and of 6.6 mmHg 6 hr after, thus maintaining a significantdecrease in the intraocular pressure. On the other hand, the Rescula eyedrops group showed an intraocular pressure decrease profile mostlysimilar to that of the compound F group (FIG. 10).

As for the local irritant effect on the eye, conjunctival edema and eyeclosure were observed 1 hr after the instillation in some animals of thecompound F group, but the animals recovered later. In the Rescula eyedrops group, conjunctival edema and eye closure in some animals wereobserved 1 hr after the instillation and lasted for up to 2 hr after theadministration. The animals recovered later.

Therefore, the compound F of the present invention shows an intraocularpressure lowering effect and a local irritant effect on the eyeequivalent to those of Rescula eye drops, and is useful as a therapeuticagent for glaucoma and high intraocular pressure.

Example 32 Prophylactic Effect on Concanavalin A-Induced Hepatitis Modelin Mice

The prophylactic effect of compound F on hepatitis was investigatedusing concanavalin A (hereinafter Con A)-induced hepatitis model. Thismodel is a hepatitis model in which parenchymal hepatocytes are injuredin a T cell-dependent manner, and exhibits similar clinical pathology toautoimmune hepatitis or fulminant hepatitis (see: References N, O, andP).

BALB/c mice (female, 7 weeks old, Japan SLC) were purchased, acclimatedfor 1 week and used for the study. Except the no-induction group, Con A(type IV, Sigma-Aldrich) dissolved in saline was administered from thetail vein of the mice at a dose of 12.5 mg/10 mL/kg to induce hepatitis.Twenty hr later, the mice were subjected to laparotomy under etheranesthesia. A 0.5 mL of blood sample was collected from the caudal venacava and heparinized plasma was obtained to measure plasma ALT and ASTactivities. Test substances were compound F (1 mg/10 mL solution),prednisolone (Nacalai Tesque, 5 mg/10 mL suspension (with 0.5 w/v %methylcellulose)) and water for injection (administered to the controlgroup), which were orally administered at a volume of 10 mL/kg 1 hrbefore Con A injection. Seven to 9 animals were used for each group.After logarithmic transformation of the data, one-way analysis ofvariance was performed for statistical evaluation.

As a result, plasma ALT and plasma AST activities of the control groupremarkably increased as compared to those of the no-induction group.Compound F and prednisolone significantly and strongly suppressed theincrease. FIG. 11 shows the data of plasma ALT.

Therefore, compound F is effective for T cell activation associatedhepatic injury.

REFERENCES

-   N) Eur. J. Immunol. 28(12): 4105-4113 (1998).-   O) Proc. Natl. Acad. Sci. 97(10): 5498-5503 (2000).-   P) J. Exp. Med. 191(1): 105-114 (2000).

INDUSTRIAL APPLICABILITY

Compound (1) of the present invention is useful as an active ingredientof medicaments. A medicament containing compound (1) of the presentinvention as an active ingredient is useful for immune diseases,diseases of the digestive tract, cardiovascular diseases, cardiacdiseases, respiratory diseases, neurological diseases, ophthalmicdiseases, renal diseases, hepatic diseases, bone diseases, skin diseasesand the like, each involving EP4. Particularly, it is useful as amedicament for the prophylaxis or treatment of ulcerative colitis,Crohn's disease, gastritis or gastric ulcer, small intestinal ulcer,nephritis, glaucoma or hepatitis.

1. A method of treating a disease involving EP4 in a subject, comprisingadministrating to the subject an effective amount of a compoundrepresented by the formula (1):

wherein R¹ and R² are each independently a hydrogen atom or a straightchain alkyl group having a carbon number of 1 to 3, and R³ is a hydrogenatom, an alkyl group having a carbon number of 1 to 4, an alkoxyalkylgroup, an aryl group, a halogen atom or a haloalkyl group, or apharmaceutically acceptable salt thereof as an active ingredient,thereby treating a disease involving EP4 in a subject, wherein thedisease whose symptoms can be mitigated by a selective EP4 agonistaction is an immune disease, a cardiovascular disease, a cardiacdisease, a skin disease or a disease selected from the group consistingof calvities, alopecia, cervical ripening failure and a hearingdisorder.
 2. The method of claim 1, wherein R¹ is a methyl group, or apharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein R³ is a methyl group, or a pharmaceutically acceptable saltthereof.
 4. The method of claim 1, wherein R² is a hydrogen atom, or apharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein R¹ is a methyl group, and R² is a hydrogen atom, or apharmaceutically acceptable salt thereof.
 6. The method of claim 1,wherein R³ is an m-methyl group, or a pharmaceutically acceptable saltthereof.
 7. The method of claim 1, wherein R¹ is a methyl group, R² is ahydrogen atom, and R³ is a methyl group, or a pharmaceuticallyacceptable salt thereof.
 8. The method of claim 1, wherein R¹ is ahydrogen atom, R² is a methyl group, and R³ is a methyl group, or apharmaceutically acceptable salt thereof.
 9. The method of claim 1,which is4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4RS)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane,4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-1-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane,4-[(Z)-(1S,5R,6R,7R)-6-[(1E,3R,4R)-3-hydroxy-4-(m-tolyl)-pentenyl]-7-hydroxy-2-oxa-4,4-difluoro-bicyclo[3.3.0]octan-3-ylidene]-1-(tetrazol-5-yl)butane,or a pharmaceutically acceptable salt thereof.
 10. The method accordingto claim 1, wherein the immune disease is amyotrophic lateral sclerosis,multiple sclerosis, Sjogren's syndrome, rheumatoid arthritis, systemiclupus erythematosus, post-transplantation rejection, arthritis, systemicinflammation response syndrome, sepsis, hemophagocytic syndrome,macrophage activation syndrome, Still's disease, Kawasaki disease,hypercytokinemia at dialysis, multiple organ failure, shock orpsoriasis.
 11. The method according to claim 1, wherein thecardiovascular disease or cardiac disease is arteriosclerosis, anginapectoris, myocardial infarction, brain disorder caused by cerebralhemorrhage, brain disorder caused by cerebral infarction, brain disordercaused by subarachnoid hemorrhage, pulmonary arterial hypertension,peripheral arterial obstruction or a symptom attributed to peripheralcirculatory disturbance.
 12. The method according to claim 1, whereinthe skin disease is Pressure ulcer or wound.
 13. The method according toclaim 1, wherein the disease involving EP4 is a disease selected fromthe group consisting of calvities, alopecia, cervical ripening failureand a hearing disorder.